CN111850123A - PCR reagent for detecting gene expression of cell angiogenesis signal pathway and application thereof - Google Patents

PCR reagent for detecting gene expression of cell angiogenesis signal pathway and application thereof Download PDF

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CN111850123A
CN111850123A CN202010666819.7A CN202010666819A CN111850123A CN 111850123 A CN111850123 A CN 111850123A CN 202010666819 A CN202010666819 A CN 202010666819A CN 111850123 A CN111850123 A CN 111850123A
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gene
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pcr reaction
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刘世海
贺桂芳
刘畅畅
蔡铎
潘华政
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Affiliated Hospital of University of Qingdao
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Abstract

The PCR reagent comprises PCR reaction primers for detecting related genes such as AKT1 gene, ANG gene, ANGPT1 gene, ANGPT2 gene and the like and reference genes and the like. The invention concentrates the angiogenesis signal molecules on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, compares the human liver cancer Huh7 with normal liver cells, discusses the possible ways of the angiogenesis signal pathway in the human liver cancer and the normal liver cells, and provides the most direct evidence for researching the regulation and control of key proteins; the invention quickly and accurately finds out the related molecules of the angiogenesis signal pathway from the transcription level, and provides a powerful tool for mechanism discussion of new targeted drugs, development of angiogenesis related inhibitors and the like.

Description

PCR reagent for detecting gene expression of cell angiogenesis signal pathway and application thereof
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a PCR reagent for detecting gene expression of a cell angiogenesis signal pathway and application thereof.
Background
An angiogenesis signal pathway is an extremely important signal transduction pathway existing in a living body, and in recent years, one of the great advances in tumor research is to establish the important role of tumor angiogenesis in tumor development and the significance of anti-angiogenesis drug therapy. Attempts have been made to kill cancer cells by cutting off the blood supply to the tumor. Since in normal adults almost all angiogenesis is pathological except for wound healing and the reproductive cycle, selective inhibition of angiogenesis has its particular advantage for tumor therapy.
Tumor angiogenesis is similar to normal angiogenesis, but is characterized by the following aspects due to the special microenvironment of the tumor: one is the loss of control. The tumor neovascularization appears rapidly, grows rapidly and is persistent; secondly, the immaturity. The tumor neovascular lumen is irregular and expanded in a sinus shape, and the wall is thin; thirdly, the tumor vascular system is highly disordered, and the blood vessels are distorted and expanded; fourthly, the micro-vessels in the tumor are abundant; fifth, the ultrastructure of the tumor vessels is abnormal, and the vessel walls have a large number of gaps and extensive inter-endothelial connections. It is the use of these specialities to study clinical therapies directed specifically at tumor vessels.
The formation of new blood vessels depends on the balance between the angiogenic growth factor and the inhibitory factor. In normal tissue, the switch for angiogenesis is in the off state due to the absence of angiogenic growth factors or under strict control by high levels of angiogenesis inhibitors, but in tumor tissue, the balance is altered by the underexpression of growth factors, which results in the tumor angiogenesis remaining in the on state, thereby causing the tumor angiogenesis to overgrow. Therefore, it is important to find a novel anti-tumor angiogenesis drug and to investigate key molecules of angiogenesis.
Disclosure of Invention
Aiming at the difficulties of determining the regulation and control of angiogenesis core signal molecules and explaining the change of the core molecules in tumor cells in the prior art, the invention provides a PCR reagent for detecting the gene expression of a cell angiogenesis signal pathway and application thereof.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a reagent for detecting an angiogenic signaling pathway, the PCR reagent comprising the following primers:
(1) the PCR reaction primers for detecting the AKT1 gene have the following primer sequences:
5'-GGTGAAGGAGAAGGCCACA-3'(SEQ ID NO:1)；
5'-TACTTCAGGGCCGTCAGG-3'(SEQ ID NO:2)；
(2) the PCR reaction primer for detecting ANG gene has the following primer sequence:
5'-CTCCATGCCAGTACCGAG-3'(SEQ ID NO:3)；
5'-GACGACGGAAAATTGACTGA-3'(SEQ ID NO:4)；
(3) the PCR reaction primers for detecting the ANGPT1 gene have the following primer sequences:
5'-CAGTACAACACAAACGCTC-3'(SEQ ID NO:5)；
5'-CTCCGACTTCATGTTTTCCA-3'(SEQ ID NO:6)；
(4) the PCR reaction primers for detecting the ANGPT2 gene have the following primer sequences:
5'-ACTTTCGGAAGAGCATGGA-3'(SEQ ID NO:7)；
5'-GAGTCATCGTATTCGAGCG-3'(SEQ ID NO:8)；
(5) the PCR reaction primer for detecting the ANGPTL4 gene has the following primer sequence:
5'-TCCACCGACCTCCCGTT-3'(SEQ ID NO:9)；
5'-CCTCATGGTCTAGGTGCTTG-3'(SEQ ID NO:10)；
(6) the PCR reaction primer for detecting the ANPEP gene has the following primer sequence:
5'-TCAACATCACGCTTATCCACC-3'(SEQ ID NO:11)；
5'-GTCGAACTCACTGACAATGAAG-3'(SEQ ID NO:12)；
(7) the PCR reaction primer for detecting the BAI1 gene has the following primer sequence:
5'-CAACCTGGTTCTCAGCATCC-3'(SEQ ID NO:13)；
5'-GACGGTCGTGTTCCTCTG-3'(SEQ ID NO:14)；
(8) the PCR primers for detecting the CCL11 gene have the following primer sequences:
5'-CCCTTCAGCGACTAGAGAG-3'(SEQ ID NO:15)；
5'-CTTGGGGTCGGCACAGAT-3'(SEQ ID NO:16)；
(9) the PCR primers for detecting the CCL2 gene have the following primer sequences:
5'-CAGCCAGATGCAATCAATGC-3'(SEQ ID NO:17)；
5'-GGAATCCTGAACCCACTTCT-3'(SEQ ID NO:18)；
(10) the primer sequence of the PCR reaction primer for detecting the CDH5 gene is as follows:
5'-TTCACGCATCGGTTGTTCAA-3'(SEQ ID NO:19)；
5'-GCTTCCACCACGATCTCATA-3'(SEQ ID NO:20)；
(11) the PCR reaction primers for detecting the COL18A1 gene have the following primer sequences:
5'-GGTCTACGTGTCGGAGCA-3'(SEQ ID NO:21)；
5'-GCCTCGTTCGCCCTTAGA-3'(SEQ ID NO:22)；
(12) the PCR reaction primers for detecting the COL4A3 gene have the following primer sequences:
5'-CAGTGGTTCTAAGGGTGAGC-3'(SEQ ID NO:23)；
5'-AAAGCCAAAGAATCCCGGAG-3'(SEQ ID NO:24)；
(13) the PCR reaction primer for detecting the CTGF gene has the following primer sequence:
5'-AAAGTGCATCCGTACTCCCA-3'(SEQ ID NO:25)；
5'-CGTCGGTACATACTCCACAG-3'(SEQ ID NO:26)；
(14) PCR primers for detecting CXCL1 gene have the following primer sequences:
5'-TGTGAAGGCAGGGGAATGT-3'(SEQ ID NO:27)；
5'-GCCCCTTTGTTCTAAGCCA-3'(SEQ ID NO:28)；
(15) PCR primers for detecting CXCL10 gene have the following primer sequences:
5'-TGGCATTCAAGGAGTACCTC-3'(SEQ ID NO:29)；
5'-GATGGCCTTCGATTCTGGATT-3'(SEQ ID NO:30)；
(16) PCR primers for detecting CXCL5 gene have the following primer sequences:
5'-GCTGCGTTGCGTTTGTTTAC-3'(SEQ ID NO:31)；
5'-GGCGAACACTTGCAGATTAC-3'(SEQ ID NO:32)；
(17) PCR primers for detecting CXCL6 gene have the following primer sequences:
5'-GAGCTGCGTTGCACTTGTT-3'(SEQ ID NO:33)；
5'-CAGTTTACCAATCGTTTTGGGG-3'(SEQ ID NO:34)；
(18) PCR primers for detecting CXCL9 gene have the following primer sequences:
5'-CAGTAGTGAGAAAGGGTCGC-3'(SEQ ID NO:35)；
5'-GGGCTTGGGGCAAATTGTT-3'(SEQ ID NO:36)；
(19) the PCR reaction primers for detecting the EDN1 gene have the following primer sequences:
5'-AGGCAACAGACCGTGAAAAT-3'(SEQ ID NO:37)；
5'-GACCTGGTTTGTCTTAGGTG-3'(SEQ ID NO:38)；
(20) the PCR reaction primer for detecting the EFNA1 gene has the following primer sequence:
5'-CAGGCCCATGACAATCCAC-3'(SEQ ID NO:39)；
5'-TGACCGATGCTATGTAGAACC-3'(SEQ ID NO:40)；
(21) the PCR reaction primers for detecting the EFNB2 gene have the following primer sequences:
5'-TCAGCCCTAACCTCTGGGG-3'(SEQ ID NO:41)；
5'-CTCCAAAGACCCATTTGATGTA-3'(SEQ ID NO:42)；
(22) the PCR reaction primer for detecting EGF gene has the following primer sequence:
5'-CCTCACCCGATAATGGTGGA-3'(SEQ ID NO:43)；
5'-CAGGAAAGCAATCACATTCCC-3'(SEQ ID NO:44)；
(23) the PCR reaction primer for detecting the ENG gene has the following primer sequence:
5'-CATCCTTCGTGGAGCTACC-3'(SEQ ID NO:45)；
5'-AGGAGTGGTCTGGATCGG-3'(SEQ ID NO:46)；
(24) the PCR reaction primers for detecting the EPHB4 gene have the following primer sequences:
5'-TGTGAACCTGACTCGATTCC-3'(SEQ ID NO:47)；
5'-TCGGCACTTGGTGTTCCC-3'(SEQ ID NO:48)；
(25) the PCR reaction primers for detecting the ERBB2 gene have the following primer sequences:
5'-CTCATCGCTCACAACCAAGT-3'(SEQ ID NO:49)；
5'-ACAGGGGTGGTATTGTTCAG-3'(SEQ ID NO:50)；
(26) the PCR reaction primers for detecting the F3 gene have the following primer sequences:
5'-GAAGCAGACGTACTTGGCAC-3'(SEQ ID NO:51)；
5'-GGGAGTTCTCATACAGAGGC-3'(SEQ ID NO:52)；
(27) the PCR reaction primers for detecting the FGF1 gene have the following primer sequences:
5'-CACCGACGGGCTTTTATACG-3'(SEQ ID NO:53)；
5'-CCATTCTTCTTGAGGCCAAC-3'(SEQ ID NO:54)；
(28) the PCR reaction primers for detecting the FGF2 gene have the following primer sequences:
5'-GTGTGTGCTAACCGTTACCT-3'(SEQ ID NO:55)；
5'-CTGCCCAGTTCGTTTCAGTG-3'(SEQ ID NO:56)；
(29) the primer sequence of the PCR reaction primer for detecting the FGFR3 gene is as follows:
5'-CCAAGCCTGTCACCGTAG-3'(SEQ ID NO:57)；
5'-AGAAACTCCCGCAGGTTACC-3'(SEQ ID NO:58)；
(30) The PCR reaction primer for detecting the FIGF gene has the following primer sequence:
5'-CTCAGTGCAGCCCTAGAGAA-3'(SEQ ID NO:59)；
5'-AACACGTTCACACAAGGGG-3'(SEQ ID NO:60)；
(31) PCR reaction primers for detecting FLT1 gene have the following primer sequences:
5'-AAAACGCATAATCTGGGACAGT-3'(SEQ ID NO:61)；
5'-CGTGGTGTGCTTATTTGGA-3'(SEQ ID NO:62)；
(32) the primer sequence of the PCR reaction primer for detecting the FN1 gene is as follows:
5'-AGAATAAGCTGTACCATCGCAA-3'(SEQ ID NO:63)；
5'-GACCACATAGGAAGTCCCAG-3'(SEQ ID NO:64)；
(33) the PCR reaction primer for detecting HGF gene has the following primer sequence:
5'-CTATCGGGGTAAAGACCTACA-3'(SEQ ID NO:65)；
5'-GTAGCGTACCTCTGGATTGC-3'(SEQ ID NO:66)；
(34) the primer sequence of the PCR reaction primer for detecting the HIF1A gene is as follows:
5'-ACCACAGGACAGTACAGGAT-3'(SEQ ID NO:67)；
5'-CGTGCTGAATAATACCACTCAC-3'(SEQ ID NO:68)；
(35) the PCR reaction primer for detecting the HPSE gene has the following primer sequence:
5'-AAGGTGTTAATGGCAAGCGT-3'(SEQ ID NO:69)；
5'-AAAGGATAGGGTAACCGCAAG-3'(SEQ ID NO:70)；
(36) the PCR reaction primers for detecting the ID1 gene have the following primer sequences:
5'-TGCTCTACGACATGAACGG-3'(SEQ ID NO:71)；
5'-AAGGTCCCTGATGTAGTCGAT-3'(SEQ ID NO:72)；
(37) the PCR reaction primers for detecting the IFNA1 gene have the following primer sequences:
5'-CCTCGCCCTTTGCTTTACT-3'(SEQ ID NO:73)；
5'-CTGTGGGTCTCAGGGAGATC-3'(SEQ ID NO:74)；
(38) the PCR reaction primer for detecting the IFNG gene has the following primer sequence:
5'-CGGTAACTGACTTGAATGTCCA-3'(SEQ ID NO:75)；
5'-CGCTTCCCTGTTTTAGCTGC-3'(SEQ ID NO:76)；
(39) the PCR reaction primer for detecting IGF1 gene has the following primer sequence:
5'-AAGATGCTCACCATGTC-3'(SEQ ID NO:77)；
5'-AGGGGCTTTTATTTCAACA-3'(SEQ ID NO:78)；
(40) the PCR reaction primers for detecting the IL1B gene have the following primer sequences:
5'-TCGACACATGGGATAACGAGG-3'(SEQ ID NO:79)；
5'-TTTTTGCTGTGAGTCCCGGAG-3'(SEQ ID NO:80)；
(41) the primer sequence of the PCR reaction primer for detecting the IL6 gene is as follows:
5'-CTGAACCTTCCAAAGATGGC-3'(SEQ ID NO:81)；
5'-TCACCAGGCAAGTCTCCTCA-3'(SEQ ID NO:82)；
(42) the primer sequence of the PCR reaction primer for detecting the IL8 gene is as follows:
5'-TTTGCCAAGGAGTGCTAAAGA-3'(SEQ ID NO:83)；
5'-ACCCTCTGCACCCAGTTTTC-3'(SEQ ID NO:84)；
(43) the PCR reaction primers for detecting the ITGAV gene have the following primer sequences:
5'-CCCATTGTACCATTGGAGAAC-3'(SEQ ID NO:85)；
5'-GGAGCATACTCAACAGTCTTTG-3'(SEQ ID NO:86)；
(44) the primer sequence of the PCR reaction primer for detecting the ITGB3 gene is as follows:
5'-GTGACCTGAAGGAGAATCTGC-3'(SEQ ID NO:87)；
5'-CGGAGTGCAATCCTCTGG-3'(SEQ ID NO:88)；
(45) the primer sequence of the PCR reaction primer for detecting the JAG1 gene is as follows:
5'-CTCCAGCCGGTGAAGACA-3'(SEQ ID NO:89)；
5'-CCTGGTGACATCATCTCCTTGT-3'(SEQ ID NO:90)；
(46) The PCR reaction primers for detecting the KDR gene have the following primer sequences:
5'-ACGTGTCACTTTGTGCAAGA-3'(SEQ ID NO:91)；
5'-TCCATGAGACGGACTCAGAA-3'(SEQ ID NO:92)；
(47) the PCR reaction primers for detecting the LECT1 gene have the following primer sequences:
5'-CATGACATTCGACCCTAGAC-3'(SEQ ID NO:93)；
5'-CAGCTACATGGCATGATGAC-3'(SEQ ID NO:94)；
(48) the PCR reaction primers for detecting the LEP gene have the following primer sequences:
5'-GCCTTCCAGAAACGTGATCC-3'(SEQ ID NO:95)；
5'-TCTGTGGAGTAGCCTGAAGC-3'(SEQ ID NO:96)；
(49) the PCR reaction primer for detecting the MDK gene has the following primer sequence:
5'-GCGGTCGCCAAAAAGAAAG-3'(SEQ ID NO:97)；
5'-ACTTGCAGTCGGCTCCAAAC-3'(SEQ ID NO:98)；
(50) the PCR reaction primer for detecting MMP14 gene has the following primer sequence:
5'-ATCTGTGACGGGAACTTTGA-3'(SEQ ID NO:99)；
5'-GCAGTGTTGATGGACGCA-3'(SEQ ID NO:100)；
(51) the PCR reaction primer for detecting MMP2 gene has the following primer sequence:
5'-ATACCCCTTTGACGGTAAGGA-3'(SEQ ID NO:101)；
5'-CCTTCTCCCAAGGTCCATAG-3'(SEQ ID NO:102)；
(52) the PCR reaction primer for detecting MMP9 gene has the following primer sequence:
5'-GACGGGTATCCCTTCGACG-3'(SEQ ID NO:103)；
5'-AAACCGAGTTGGAACCACGA-3'(SEQ ID NO:104)；
(53) the primer sequence of the PCR reaction primer for detecting the NOS3 gene is as follows:
5'-CGCACTTCTGTGCCTTTGCTC-3'(SEQ ID NO:105)；
5'-GCTCGGGTGGATTTGCTGCTC-3'(SEQ ID NO:106)；
(54) the primer sequence of the PCR reaction primer for detecting the NOTCH4 gene is as follows:
5'-TAGGGGCTCTTCTCGTCCT-3'(SEQ ID NO:107)；
5'-AACTTCTGCCTTTGGCTTC-3'(SEQ ID NO:108)；
(55) the PCR reaction primers for detecting the NRP1 gene have the following primer sequences:
5'-CGTGGAAGTCTTCGATGGAG-3'(SEQ ID NO:109)；
5'-ACCATGTGTTTCGTAGTCAGA-3'(SEQ ID NO:110)；
(56) the PCR reaction primers for detecting the NRP2 gene have the following primer sequences:
5'-CAACGGGACCATCGAATCTC-3'(SEQ ID NO:111)；
5'-CAGCCAATCGTACTTGCAGT-3'(SEQ ID NO:112)；
(57) the PCR reaction primers for detecting the PDGFA gene have the following primer sequences:
5'-GCAAGACCAGGACGGTCATT-3'(SEQ ID NO:113)；
5'-GCACTTGACACTGCTCGT-3'(SEQ ID NO:114)；
(58) the PCR reaction primers for detecting the PECAM1 gene have the following primer sequences:
5'-CCGTGACGGAATCCTTCTCT-3'(SEQ ID NO:115)；
5'-CTGGACTCCACTTTGCAC-3'(SEQ ID NO:116)；
(59) the PCR reaction primers for detecting the PF4 gene have the following primer sequences:
5'-TTAACGGAGAGCCTGCTGAG-3'(SEQ ID NO:117)；
5'-GCCAGTTGCAAATGGCATTAGAAGGG-3'(SEQ ID NO:118)；
(60) the PCR reaction primers for detecting the PGF gene have the following primer sequences:
5'-AACGGCTCGTCAGAGGTG-3'(SEQ ID NO:119)；
5'-CAGTGCAGATTCTCATCGCC-3'(SEQ ID NO:120)；
(61) the PCR reaction primers for detecting the PLAU gene have the following primer sequences:
5'-CTTGTCCAAGAGTGCATGGT-3'(SEQ ID NO:121)；
5'-CAGGGCTGGTTCTCGATGG-3'(SEQ ID NO:122)；
(62) The PCR reaction primers for detecting the PLG gene have the following primer sequences:
5'-GCGACATTCTTGAGTGTGAAG-3'(SEQ ID NO:123)；
5'-CGGGGTTACGACAGTAATTCTT-3'(SEQ ID NO:124)；
(63) the PCR reaction primers for detecting the PROK2 gene have the following primer sequences:
5'-TGACAAGGACTCCCAATGTG-3'(SEQ ID NO:125)；
5'-GACCCAGATACTGACAGCAC-3'(SEQ ID NO:126)；
(64) the PCR reaction primers for detecting the PTGS1 gene have the following primer sequences:
5'-TCTGTGCCTAAAGATTGCCC-3'(SEQ ID NO:127)；
5'-TCTCCATAAATGTGGCCGAG-3'(SEQ ID NO:128)；
(65) the PCR reaction primers for detecting the S1PR1 gene have the following primer sequences:
5'-CTGCTGGCAAATTCAAGCGA-3'(SEQ ID NO:129)；
5'-TTGTCCCCTTCGTCTTTCTG-3'(SEQ ID NO:130)；
(66) the PCR reaction primer for detecting the SERPINE1 gene has the following primer sequence:
5'-GTGGACTTTTCAGAGGTGGA-3'(SEQ ID NO:131)；
5'-GCCGTTGAAGTAGAGGGCATT-3'(SEQ ID NO:132)；
(67) the PCR reaction primers for detecting the SERPINF1 gene have the following primer sequences:
5'-GAAATTCCCGATGAGATCAGC-3'(SEQ ID NO:133)；
5'-GTCAAACTTTGTTACCCACTGC-3'(SEQ ID NO:134)；
(68) the PCR reaction primers for detecting the SPHK1 gene have the following primer sequences:
5'-GAGTGGGTTCCAAGACACCT-3'(SEQ ID NO:135)；
5'-GGTGCAGCAAACATCTCAC-3'(SEQ ID NO:136)；
(69) the PCR reaction primers for detecting the TEK gene have the following primer sequences:
5'-AGGATACGAACCATGAAGATGC-3'(SEQ ID NO:137)；
5'-GGGCACTGAATGGATGAAG-3'(SEQ ID NO:138)；
(70) the PCR reaction primers for detecting the TGFA gene have the following primer sequences:
5'-GGTCCGAAAACACTGTGAGT-3'(SEQ ID NO:139)；
5'-GCAAGCGGTTCTTCCCTTC-3'(SEQ ID NO:140)；
(71) the PCR reaction primers for detecting the TGFB1 gene have the following primer sequences:
5'-AATTCCTGGCGATACCTCAG-3'(SEQ ID NO:141)；
5'-CACAACTCCGGTGACATCAA-3'(SEQ ID NO:142)；
(72) the PCR reaction primers for detecting the TGFB2 gene have the following primer sequences:
5'-CATCCCGCCCACTTTCTAC-3'(SEQ ID NO:143)；
5'-GCTCAATCCGTTGTTCAGGC-3'(SEQ ID NO:144)；
(73) the PCR reaction primers for detecting the TGFBR1 gene have the following primer sequences:
5'-CGGCGTTACAGTGTTTCTG-3'(SEQ ID NO:145)；
5'-GCACATACAAACGGCCTATCT-3'(SEQ ID NO:146)；
(74) the PCR reaction primers for detecting the THBS1 gene have the following primer sequences:
5'-AGACTCCGCATCGCAAAG-3'(SEQ ID NO:147)；
5'-CACCACGTTGTTGTCAAGGG-3'(SEQ ID NO:148)；
(75) the PCR reaction primers for detecting the THBS2 gene have the following primer sequences:
5'-CCCTCCTAAGACAAGGAACA-3'(SEQ ID NO:149)；
5'-CACCCACGTTTCATTTTCCG-3'(SEQ ID NO:150)；
(76) the primer sequence of the PCR reaction primer for detecting the TIE1 gene is as follows:
5'-ATTGCCGACTCCAGTGCC-3'(SEQ ID NO:151)；
5'-TCTGGCTTGCGTAGCTCT-3'(SEQ ID NO:152)；
(77) the primer sequence of the PCR reaction primer for detecting the TIMP1 gene is as follows:
5'-CCACCTTATACCAGCGTTATGA-3'(SEQ ID NO:153)；
5'-GTGTAGACGAACCGGATGTC-3'(SEQ ID NO:154)；
(78) The primer sequence of the PCR reaction primer for detecting the TIMP2 gene is as follows:
5'-CTGCGAGTGCAAGATCAC-3'(SEQ ID NO:155)；
5'-GGTGCCCGTTGATGTTCTTC-3'(SEQ ID NO:156)；
(79) the primer sequence of the PCR reaction primer for detecting the TIMP3 gene is as follows:
5'-TGCAACTTCGTGGAGAGGTG-3'(SEQ ID NO:157)；
5'-AGGTGATACCGATAGTTCAGC-3'(SEQ ID NO:158)；
(80) the PCR reaction primer for detecting TNF gene has the following primer sequence:
5'-CTCTCTCTAATCAGCCCTCTG-3'(SEQ ID NO:159)；
5'-GAGGACCTGGGAGTAGATGAG-3'(SEQ ID NO:160)；
(81) the PCR reaction primer for detecting the TYMP gene has the following primer sequence:
5'-GTGTGGGTGACAAGGTCAG-3'(SEQ ID NO:161)；
5'-CAGCACTTGCATCTGCTC-3'(SEQ ID NO:162)；
(82) the PCR reaction primers for detecting the VEGFA gene have the following primer sequences:
5'-CAACATCACCATGCAGATTATGC-3'(SEQ ID NO:163)；
5'-TCGGCTTGTCACATTTTTCTTG-3'(SEQ ID NO:164)；
(83) the PCR reaction primers for detecting the VEGFB gene have the following primer sequences:
5'-AGATGTCCCTGGAAGAACACA-3'(SEQ ID NO:165)；
5'-AGTGGGATGGGTGATGTCAG-3'(SEQ ID NO:166)；
(84) the PCR reaction primers for detecting the VEGFC gene have the following primer sequences:
5'-TGTGTGTCCGTCTACAGATGT-3'(SEQ ID NO:167)；
5'-GAAGTGTGATTGGCAAAACTGA-3'(SEQ ID NO:168)；
(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:
5'-GAGCTACGAGCTGCCTGAC-3'(SEQ ID NO:169)；
5'-GTAGTTTCGTGGATGCCACA-3'(SEQ ID NO:170)；
(86) the PCR reaction primers for detecting the B2M gene have the following primer sequences:
5'-TTAGCTGTGCTCGCGCTACTC-3'(SEQ ID NO:171)；
5'-TGGTTCACACGGCAGGCATAC-3'(SEQ ID NO:172)；
(87) the PCR reaction primers for detecting the GAPDH gene have the following primer sequences:
5'-AGTGACACCCACTCCTCCA-3'(SEQ ID NO:173)；
5'-TGTAGCCAAATTCGTTGTC-3'(SEQ ID NO:174)；
(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:
5'-CTCATGGACTGATTATGGACAG-3'(SEQ ID NO:175)；
5'-TCCAGCAGGTCAGCAAAGAA-3'(SEQ ID NO:176)；
(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:
5'-TAAACCCTGCGTGGCAATCCCT-3'(SEQ ID NO:177)；
5'-TGAACACAAAGCCCACATTCCC-3'(SEQ ID NO:178)。
compared with the prior art, the invention has the advantages and positive effects that: the invention provides a PCR detection reagent for detecting the change of cell angiogenesis signal channel core molecules, genes related to angiogenesis can be rapidly detected at the transcription level through real-time fluorescence quantification by means of the detection reagent, and the core molecules and action mechanisms of tumor angiogenesis signal channels are analyzed and researched on the basis, so that angiogenesis regulation signal channels of tumor-related drugs can be rapidly and accurately found, and a powerful tool is provided for anti-cancer drug screening, mechanism discussion of new targeted drugs and the like.
The invention determines 84 genes related to an angiogenesis signal path through a large number of creative experiments, and the invention focuses on the combination of primers. The primer dimer often appears in the combination of any primer during the specific PCR, which affects the failure of the target gene amplification, or the non-specific amplification, which greatly affects the effectiveness of the PCR result. Under the same experimental condition, PCR detection of 89 genes (84 of which are detection genes and 5 of which are reference genes) is carried out simultaneously, so that not only is primer design required, but also the optimal amplification condition is required to be well grasped, which is a technical difficulty. The most central is primer design, the invention obtains the best amplification condition through multiple pcr and adjustment, and ensures that the amplification efficiency of each gene is very high without nonspecific amplification.
The invention also provides a PCR method for specific detection, the experimental system can be carried out on any real-time fluorescent quantitative PCR instrument, and the PCR reaction instrument is LightCycler480 of Roche. The 87 pairs of primers were placed in a 96-well plate and subjected to real-time fluorescent quantitative PCR detection. Changes in the core molecule with respect to the angiogenic signaling pathway can be obtained by a single reaction. The method has the advantages of simple and convenient experimental operation, low cost and good result repeatability, is favorable for reducing unnecessary detection in the downstream, and is an important means for researching the action mechanism of the tumor-related medicament.
Other features and advantages of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Drawings
FIG. 1 shows that the invention utilizes angiogenesis signal pathway to screen out human liver cancer Huh7 and normal liver cell livercell differential expression genes, wherein 1: huh7 cells; 2: normal liver cells.
FIG. 2 shows real-time fluorescent quantitative PCR detection of angiogenesis signaling pathway gene expression.
FIG. 3 shows a melting curve analysis of the expression of angiogenic signaling pathway genes.
FIG. 4 shows the difference of angiogenesis signaling pathway gene expression in human liver cancer Huh7, normal liver cell lever cell.
FIG. 5 shows the validation of the results of screening the angiogenesis core molecule of liver cancer.
FIG. 6 shows the effect of core angiogenic factors on Huh7 angiogenesis.
FIG. 7 shows the effect of the core angiogenic factor on tumors detected in nude mice tumorigenesis experiments.
FIG. 8 shows the effect of CD31 immunohistochemical detection of the gene of interest on tumor angiogenesis.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
Example 1
The invention is exemplified by human liver cancer Huh7 and normal liver cell, but the invention is not limited to the two cells, and the PCR detection reagent can be applied to common cancers such as breast cancer, lung cancer, gastric cancer and the like.
The invention is characterized by culturing two cell lines: human hepatoma Huh7, normal hepatocyte liver cell and then the angiogenesis signal pathway detection reagent is used for detecting the change of the nuclear cardiovascular generation molecules in the two cell lines.
The human liver cancer Huh7, normal liver cell lever cell used in the present invention were purchased from American ATCC company, Shanghai cell bank, DMEM medium, fetal bovine serum were purchased from American Gibco company, RNAioso for extracting total RNA, reverse transcription kit,
Figure BDA0002580761060000111
the Premix Ex Taq kit was purchased from TaKaRa.
The method for detecting the angiogenesis signal pathway in the cell by using the reagent comprises the following specific steps:
first, culture and passage of human liver cancer Huh7 and normal liver cell
Taking Huh7 cells as an example, 1mL of 0.2% pancreatin solution is added into a 60mm cell culture plate, the pancreatin is gently shaken to be in full contact with the cells, the cell culture plate is incubated for 2min at 37 ℃ in a carbon dioxide incubator, after most of cells are observed to float under a mirror, 4mL of DMEM medium containing 10% FBS is added, the DMEM medium is evenly blown and added into a 15mL centrifugal tube, the 15mL centrifugal tube is centrifuged for 6min at 2000g, the supernatant is discarded, a corresponding proper volume of culture solution is added according to the cell types, and the cell is blown and beaten repeatedly by a 10mL pipette for a plurality of times to free the cells. In this process, air bubbles are prevented from being generated, so as not to influence the cell count. Taking 20 mu L of cell suspension, dripping into a cell counting plate, and counting the total number of cells in four big grids at four corners according to a cell counting method. When counting, only cells with intact cell morphology are counted, and the number of stacked cells is counted as 1 cell. The calculation formula is as follows:
Cell concentration (one/mL) ═ 4 big grid cell count × 104X dilution factor/4.
Diluting to 5 × 10 with corresponding culture solution according to cell type6Adding 2mL of cell dilution into 60mm cell culture dish, placing at 37 deg.C and 5% CO2The cells are cultured in a cell culture box, the cells are attached to the wall after 0.5h-1h generally, the cells begin to grow after a plurality of hours, and the cells become monolayer cells after 24h-48 h.
Second, extraction of Total RNA
1) Taking CO2After sucking out the culture medium from a disk of cells in a 60mm plate in logarithmic phase in an incubator, washing the disk of cells for 2 times by using sterile 1 XPBS, adding 1mL of RNAasso on the surfaces of the cells, repeatedly sucking the cells uniformly by using a gun head, and transferring the cells into a sterile tube.
2) Incubate at room temperature for 5min to solubilize the nucleoprotein. Add 0.2mL phenol/chloroform (1:1), shake vigorously for 15sec, incubate at room temperature for 2-3 min.
3) Cells were stratified by centrifugation at 12000g for 15min at 4 ℃.
4) Transferring the upper layer liquid to a centrifuge tube without RNase, adding 0.5mL isopropanol, reversing and mixing uniformly for 3 times, and incubating for 10min at room temperature.
5) Centrifugation at 12000g for 10min at 4 ℃ resulted in the formation of a bottom pellet of RNA.
6) The supernatant was discarded and the pellet was washed with 1mL of RNase-free 75% ethanol. Centrifuge at 7000g for 5min at 4 ℃.
8) The RNA was air dried for about 5-10 min. It should not be dried too much.
9) RNA was dissolved in 30-50. mu.L DEPC water.
The results are shown in FIG. 1, and it can be seen from the electrophoresis in FIG. 1 that lane 1 is Huh7, lane 2 is normal liver cell, the total RNA band is complete, and it is suitable for downstream reverse transcription and other experiments.
Synthesis of cDNA
1) The following reaction mixture was added to a pre-cooled tube:
1-5 mug of total RNA; oligo (dT) (0.5. mu.g/. mu.l) 1. mu.L; the volume was adjusted to 12. mu.L with RNase-free water.
2) Mixing, and centrifuging for 3-5 sec.
3) Acting at 70 deg.C for 5min, ice-cooling for 30sec, and centrifuging for 3-5 sec.
4) The tube was ice-cooled and the following components were added:
5×Reaction buffer 4μL；
Rnase Inhibiter(20U/μL)1μL；
dNTP mix(10mM)2μL；
5) mix gently and centrifuge for 3-5 sec.
6) After being bathed at 37 ℃ for 5min, 1. mu.L of M-MuLV Reverse Transcriptase (20U/. mu.L) was added and mixed well.
7) Acting at 37 deg.C for 60 min. The reaction was terminated by incubation at 70 ℃ for 10min and downstream testing was performed on ice.
Four, real-time fluorescent quantitative PCR
Taking the synthesized cDNA as a template, and carrying out real-time fluorescence quantitative PCR reaction according to the following system:
Figure BDA0002580761060000121
premix Ex Taq 10. mu.L; upstream primer 0.1-0.2 μmol; downstream primer 0.1-0.2 μmol; 25-100ng of cDNA template; DW made up the volume to 20. mu.L.
The following program was set up on the LightCycler480 instrument from roche: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
The upstream and downstream primers in the PCR reaction are respectively the PCR reagents (including 84 primers of the detection genes and 5 primers of the reference genes) described in the invention, and the sequences are shown as SEQ ID NO:1-SEQ ID NO: 178.
And (3) placing the primers on a 96-well plate to perform PCR reaction, wherein each pair of primers is placed in any one well, and 89 pairs of primers are placed in 89 wells in total. FIG. 2 shows the results of real-time fluorescence quantitative PCR amplification of human hepatoma Huh7 and normal hepatocyte liver cell, and the amplification curve shows that the target gene has good amplification and is suitable for downstream Ct value analysis.
Fifth, data analysis
And calculating the expression difference of the genes related to the core angiogenesis by using the Ct value of each gene.
FIG. 3 is a dissolution curve analysis of human hepatoma Huh7 and normal hepatocyte liver cell after real-time fluorescent quantitative PCR, from the dissolution curve, the peak shape of the target gene is single, the amplification product is the target gene, and there are no phenomena of primer dimer, non-specific amplification, etc., which shows that the primer and PCR system are good and suitable for downstream experiments.
FIG. 4 shows the results of differential expression analysis after real-time fluorescent quantitative PCR of human hepatoma Huh7 and normal hepatocyte liver cell, from which angiogenic signaling pathway molecules closely related to the occurrence and development of human hepatoma can be screened out. The map shows that the expression of molecules such as PECAM1, SERPINE1, MMP9 and the like is obviously up-regulated, and the genes are preliminarily shown to be closely related to the generation of tumor vessels. The PECAM1 is called Platelet-endothelial cell adhesion molecule (PECAM 1), also known as CD31, has a molecular weight of 130kDa, belongs to immunoglobulin superfamily members in structure, participates in leukocyte migration, angiogenesis and integrin activation, which is highly consistent with the screening purpose of the kit, and fully proves the effectiveness of the kit screening. Many other cell types within the tumor microenvironment also contribute to angiogenesis. Neutrophils constitute a large part of the immune cell infiltrate and can promote tumor angiogenesis by a variety of mechanisms. This involves the release of MMPs into the tumor microenvironment, triggering the release of VEGF and other angiogenic factors. Similarly, other immune cell types (e.g., B cells and T cells) secrete VEGF-A, MMP9, etc., which indirectly affects angiogenesis. The kit screens MMP9 closely related to liver cancer angiogenesis by implementing fluorescent quantitative PCR, and lays a foundation for researching the functions of downstream tumor vessels.
Sixth, Re-validation of pathway molecules
The screened results are verified again by adopting real-time fluorescent quantitative PCR, the results are shown in figure 5, and the results in figure 5 prove that the expressions of molecules such as COL4A3, CXCL6, TGFB1, PECAM1, SERPINE1, MMP9, CTGF, LECT1 and the like are all obviously up-regulated and are closely related to angiogenesis molecules of human liver cancer, and the reliability of the screening results is further verified.
The cascade of malignant tumor metastasis involves a series of steps including local infiltration, infiltration of tumor cells into blood vessels, circulation of tumor cells within blood and lymph vessels, adhesion to distant sites, extravasation, and eventually the formation of new tumors at a distance. TGFB1 regulates many links in the process, such as promoting matrix environment formation favorable for infiltration and metastasis, promoting angiogenesis, inhibiting host anti-tumor immune response, and the like, and TGFB1 screened by the PCR reagent further proves the process.
Seventhly, functional verification of core angiogenesis molecules
The effect of CTCF, MMP9, PECAM1 and SERPINE1 on angiogenesis of hepatoma cells Huh7 was examined by an angiogenesis experiment.
1. The day before the experiment Matrigel was placed in an ice box and placed in a 4 degree freezer to allow the gel to melt slowly overnight. (Note: some 4-degree precooled tips were also prepared for aspirating Matrigel)
2. Before starting the experiment, Matrigel was kept in an ice box at all times.
3. The sterilized package is opened and the angiogenic slide is removed.
4. Mu.l Matrigel was added to each well. Note that the tip was added perpendicular to the inner bore and just above the matrix, preventing the matrix from flowing through the upper bore and leaving residual gum.
5. Cover the angiogenesis slide.
6. A10 cm petri dish was prepared and soaked in water paper towels to make a wet box.
7. The angiogenesis slide was placed into the culture dish and the dish lid was closed.
8. The whole culture dish is placed in an incubator, and is kept stand for about 30 minutes until the gel is coagulated.
9. Waiting for the cell suspension to be prepared at the same time.
10. In this experiment, a control group was included, and cells were transfected with siRNA of interest and incubated for 24 hours prior to cell suspension preparation. The next day a density of 2 x 10 was prepared5cells/ml cell suspension, mix well.
11. The angiogenesis slide with the glue solidified was removed from the wet box.
12. Add 50. mu.l of cell suspension to each well, taking care to keep the tip vertically above the upper well, without touching the gel in the lower well.
13. Also, if a sufficient amount of liquid is not added, as viewed in the grid paper, and if not, cell-free medium is added to just fill the upper well.
14. The lid was closed and left to stand, after a period of time, all the cells settled down on the surface of Matrigel.
15. And collecting images and counting results.
Under a microscope, a tunnel-like and saccular structure surrounded by tumor cells of liver cancer can be seen, and expression of CTCF, MMP9, PECAM1 or SERPINE1 is knocked down, the group of liver cancer cells all present a massive structure and cannot form a tunnel-like vascular structure through culture observation of Matrigel, so that CTCF, MMP9, PECAM1 or SERPINE1 can obviously inhibit generation of tumor vessels from results, and the detection effectiveness and reliability of the tumor angiogenesis PCR reagent are further verified.
Eighthly, adopting nude mouse tumorigenesis experiment to detect
1. The cell state is the key of nude mouse tumorigenicity experiment, so the cell growth state must be good, the cell in logarithmic growth phase is taken, and the density of the Huh7 cell is about 80-90%; each group of cells was used 24 hours after transfection with siRNA and siRNA of interest. The day night before cell collection was replaced with fresh medium.
2. Cells were trypsinized and washed twice with pre-cooled PBS in order to remove serum from the cells.
3. The cell sediment is blown by PBS or serum-free medium to a proper concentration, and the amount of the cells inoculated to the subcutaneous tumor is 2 multiplied by 10 6The inoculation volume was 0.1ml per cell.
4. After the cells are digested, the cells should be inoculated under the skin of the nude mice as soon as possible, and the inoculation is generally completed within half an hour as possible, and the cell suspension is placed on ice in the process to reduce the metabolism of the cells.
5. The selected nude mice are generally 6 weeks old and have the weight of about 18-20g, and the area with rich blood supply at the planting position is the middle and back parts of armpits.
6. Before inoculation, the cell suspension is blown off fully by a gun, so that cell agglomeration is prevented, and the cell survival rate is reduced.
7. Grouping: huh7 cell group, Huh7 transfection irrelevant siRNA group, Huh7 transfection siRNA group (6 each).
8. And taking out the mouse cages for the nude mice, and sequentially placing the mouse cages in a clean bench. The larger size mice were weighed and anesthetized with a 0.3% solution dose of sodium pentobarbital at 0.1ml/10g intraperitoneal injection. About 5 minutes, the mice began to rest and were fixed in their prone position.
9. During inoculation, the needle head is inserted a little bit deep into the subcutaneous needle, about 1cm deep, and the overflow of the cell suspension from the needle eye after injection is reduced.
10. After all cells were injected, the ear-cutting markers were clipped and the grouping was recorded.
11. The state of the mice is noticed, and whether the mice die due to surgical stimulation, infection and the like is found in time.
12. On day 24 after inoculation, mice were anesthetized, then cervical vertebrae were detached, mice sacrificed, and carcasses were dissected to observe tumor growth and whether metastases formed in the liver, lungs and other organs. Then, the pathology was performed after fixation with 10% formaldehyde solution overnight.
Tumor growth is dependent on the formation of new blood vessels. Research in recent 30 years proves that angiogenesis is a key step in the growth and metastasis processes of many tumors, and anti-angiogenesis therapy, especially anti-angiogenesis gene therapy, is used as a new field of tumor therapy, brings new hope for improving tumor prognosis, and becomes a hotspot of research. The growth of the transplanted tumor can be seen by knocking down the expression of CTCF, MMP9, PECAM1 or SERPINE1 in the transplanted tumor of the nude mouse, as shown in FIG. 7, the growth of the objective RNA knocked down grouped tumor is inhibited, and the tumor quality is obviously smaller than that of the control group. The result further proves that the tumor angiogenesis detection reagent is not only effective in-vitro (in-cell) detection, but also has obvious effect in-vivo (nude mouse tumorigenesis experiment) detection, and reflects the stability and reliability of the tumor angiogenesis PCR reagent.
Ninthly, detecting mouse tumor angiogenesis by immunohistochemistry
1. Tumors from each group of mice were routinely paraffin embedded and paraffin sectioned, and the paraffin sections were then baked overnight in an oven at 60 ℃.
2. Dewaxing in xylene, adding gradient ethanol into water (anhydrous ethanol, 95% ethanol), and soaking in distilled water.
3. Antigen retrieval
500ml of EDTA antigen retrieval working solution is taken into a 1000ml beaker and heated on a low-power electric furnace until boiling and slight boiling (in order to prevent flaking). The tissue slices were slowly placed in a beaker. Heating is continued, and the liquid is kept in a slightly boiling state for 20 minutes. The beaker was removed from the fire source, cooled naturally at room temperature and then sliced, washed with distilled water for 1 time and 3 minutes, and washed with TBS for 2 times and 3 minutes each time.
4. 50ul of 3% hydrogen peroxide solution was added to each section and incubated at room temperature for 10min to block endogenous peroxidase activity. TBS was washed 3 times for 3min each.
5. TBS was removed and 50ul primary antibody (rabbit anti-mouse CD31, diluted 1: 200) was added overnight at 4 ℃.
TBS washing was performed 3 times for 5min, TBS solution was removed, 50ul of polymer enhancer (reagent A) was added to each section, incubation was performed at room temperature for 20min, and TBS washing was performed 3 times for 3min each.
7. TBS solution was removed and 50ul of peroxidase-labeled anti-rabbit polymer (reagent B) was added to each section, incubated at room temperature for 30min and washed 3 times with TBS for 3min each.
8. TBS solution was removed and 50ul of freshly prepared DAB was added to each section and allowed to develop for 10 min.
9. Washing with tap water, hematoxylin counterstaining for 10min, and washing with water. 0.1% hydrochloric acid differentiation, tap water washing, TBS bluing.
10. Without dehydration, the neutral resin is directly used for sealing the sheet.
As a result, as shown in FIG. 8, in the tumor of the mouse in which the target gene was down-regulated, the amount of angiogenesis was significantly reduced as found by staining the blood vessels with CD31 (CD31 is a specific marker for tumor angiogenesis). Therefore, the core angiogenesis factor screened by the tumor angiogenesis PCR reagent can be proved to have important guiding significance for researching the core factor of tumor angiogenesis.
Through a series of experimental results, genes CTCF, MMP9, PECAM1 or SERPINE1 related to liver cancer angiogenesis are finally proved to have obvious regulation and control effects, and the effectiveness of the technical scheme is also proved through in vivo and in vitro experiments.
The invention concentrates the molecules closely related to the angiogenesis signal path on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, discusses the possible path of the angiogenesis signal path in tumor/normal cells, and provides the most direct evidence for researching the regulation and control of key protein; the invention can quickly and accurately find the related molecules of the angiogenesis signal pathway from the transcription level, and provides a powerful tool for screening anti-cancer drugs, discussing the mechanism of new targeted drugs and the like.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> affiliated Hospital of Qingdao university
<120> PCR reagent for detecting gene expression of cell angiogenesis signal pathway and application thereof
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<213> Artificial Sequence (Artificial Sequence)
<400>47
tgtgaacctg actcgattcc 20
<210>48
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
tcggcacttg gtgttccc 18
<210>49
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
ctcatcgctc acaaccaagt 20
<210>50
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
acaggggtgg tattgttcag 20
<210>51
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gaagcagacg tacttggcac 20
<210>52
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
gggagttctc atacagaggc 20
<210>53
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
caccgacggg cttttatacg 20
<210>54
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
ccattcttct tgaggccaac 20
<210>55
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
gtgtgtgcta accgttacct 20
<210>56
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
ctgcccagtt cgtttcagtg20
<210>57
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
ccaagcctgt caccgtag 18
<210>58
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
agaaactccc gcaggttacc 20
<210>59
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
ctcagtgcag ccctagagaa 20
<210>60
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
aacacgttca cacaagggg 19
<210>61
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
aaaacgcata atctgggaca gt 22
<210>62
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
cgtggtgtgc ttatttgga 19
<210>63
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
agaataagct gtaccatcgc aa 22
<210>64
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
gaccacatag gaagtcccag 20
<210>65
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
ctatcggggt aaagacctac a 21
<210>66
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
gtagcgtacc tctggattgc 20
<210>67
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
accacaggac agtacaggat 20
<210>68
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
cgtgctgaat aataccactc ac 22
<210>69
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
aaggtgttaa tggcaagcgt 20
<210>70
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
aaaggatagg gtaaccgcaa g21
<210>71
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
tgctctacga catgaacgg 19
<210>72
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
aaggtccctg atgtagtcga t 21
<210>73
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
cctcgccctt tgctttact 19
<210>74
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
ctgtgggtct cagggagatc 20
<210>75
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
cggtaactga cttgaatgtc ca 22
<210>76
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
cgcttccctg ttttagctgc 20
<210>77
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>77
aagatgctca ccatgtc 17
<210>78
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>78
aggggctttt atttcaaca 19
<210>79
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>79
tcgacacatg ggataacgag g 21
<210>80
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>80
tttttgctgt gagtcccgga g 21
<210>81
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>81
ctgaaccttc caaagatggc 20
<210>82
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>82
tcaccaggca agtctcctca 20
<210>83
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>83
tttgccaagg agtgctaaag a 21
<210>84
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>84
accctctgca cccagttttc20
<210>85
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>85
cccattgtac cattggagaa c 21
<210>86
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>86
ggagcatact caacagtctt tg 22
<210>87
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>87
gtgacctgaa ggagaatctg c 21
<210>88
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>88
cggagtgcaa tcctctgg 18
<210>89
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>89
ctccagccgg tgaagaca 18
<210>90
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>90
cctggtgaca tcatctcctt gt 22
<210>91
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>91
acgtgtcact ttgtgcaaga 20
<210>92
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>92
tccatgagac ggactcagaa 20
<210>93
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>93
catgacattc gaccctagac 20
<210>94
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>94
cagctacatg gcatgatgac 20
<210>95
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>95
gccttccaga aacgtgatcc 20
<210>96
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>96
tctgtggagt agcctgaagc 20
<210>97
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>97
gcggtcgcca aaaagaaag 19
<210>98
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>98
acttgcagtc ggctccaaac20
<210>99
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>99
atctgtgacg ggaactttga 20
<210>100
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>100
gcagtgttga tggacgca 18
<210>101
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>101
ataccccttt gacggtaagg a 21
<210>102
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>102
ccttctccca aggtccatag 20
<210>103
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>103
gacgggtatc ccttcgacg 19
<210>104
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>104
aaaccgagtt ggaaccacga 20
<210>105
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>105
cgcacttctg tgcctttgct c 21
<210>106
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>106
gctcgggtgg atttgctgct c 21
<210>107
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>107
taggggctct tctcgtcct 19
<210>108
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>108
aacttctgcc tttggcttc 19
<210>109
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>109
cgtggaagtc ttcgatggag 20
<210>110
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>110
accatgtgtt tcgtagtcag a 21
<210>111
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>111
caacgggacc atcgaatctc 20
<210>112
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>112
cagccaatcg tacttgcagt20
<210>113
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>113
gcaagaccag gacggtcatt 20
<210>114
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>114
gcacttgaca ctgctcgt 18
<210>115
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>115
ccgtgacgga atccttctct 20
<210>116
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>116
ctggactcca ctttgcac 18
<210>117
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>117
ttaacggaga gcctgctgag 20
<210>118
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>118
gccagttgca aatggcatta gaaggg 26
<210>119
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>119
aacggctcgt cagaggtg 18
<210>120
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>120
cagtgcagat tctcatcgcc 20
<210>121
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>121
cttgtccaag agtgcatggt 20
<210>122
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>122
cagggctggt tctcgatgg 19
<210>123
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>123
gcgacattct tgagtgtgaa g 21
<210>124
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>124
cggggttacg acagtaattc tt 22
<210>125
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>125
tgacaaggac tcccaatgtg 20
<210>126
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>126
gacccagata ctgacagcac20
<210>127
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>127
tctgtgccta aagattgccc 20
<210>128
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>128
tctccataaa tgtggccgag 20
<210>129
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>129
ctgctggcaa attcaagcga 20
<210>130
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>130
ttgtcccctt cgtctttctg 20
<210>131
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>131
gtggactttt cagaggtgga 20
<210>132
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>132
gccgttgaag tagagggcat t 21
<210>133
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>133
gaaattcccg atgagatcag c 21
<210>134
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>134
gtcaaacttt gttacccact gc 22
<210>135
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>135
gagtgggttc caagacacct 20
<210>136
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>136
ggtgcagcaa acatctcac 19
<210>137
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>137
aggatacgaa ccatgaagat gc 22
<210>138
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>138
gggcactgaa tggatgaag 19
<210>139
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>139
ggtccgaaaa cactgtgagt 20
<210>140
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>140
gcaagcggtt cttcccttc19
<210>141
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>141
aattcctggc gatacctcag 20
<210>142
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>142
cacaactccg gtgacatcaa 20
<210>143
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>143
catcccgccc actttctac 19
<210>144
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>144
gctcaatccg ttgttcagg 19
<210>145
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>145
cggcgttaca gtgtttctg 19
<210>146
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>146
gcacatacaa acggcctatc t 21
<210>147
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>147
agactccgca tcgcaaag 18
<210>148
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>148
caccacgttg ttgtcaaggg 20
<210>149
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>149
ccctcctaag acaaggaaca 20
<210>150
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>150
cacccacgtt tcattttccg 20
<210>151
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>151
attgccgact ccagtgcc 18
<210>152
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>152
tctggcttgc gtagctct 18
<210>153
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>153
ccaccttata ccagcgttat ga 22
<210>154
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>154
gtgtagacga accggatgtc 20
<210>155
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>155
ctgcgagtgc aagatcac 18
<210>156
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>156
ggtgcccgtt gatgttcttc 20
<210>157
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>157
tgcaacttcg tggagaggtg 20
<210>158
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>158
aggtgatacc gatagttcag c 21
<210>159
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>159
ctctctctaa tcagccctct g 21
<210>160
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>160
gaggacctgg gagtagatga g 21
<210>161
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>161
gtgtgggtga caaggtcag 19
<210>162
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>162
cagcacttgc atctgctc 18
<210>163
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>163
caacatcacc atgcagatta tgc 23
<210>164
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>164
tcggcttgtc acatttttct tg 22
<210>165
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>165
agatgtccct ggaagaacac a 21
<210>166
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>166
agtgggatgg gtgatgtcag 20
<210>167
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>167
tgtgtgtccg tctacagatg t 21
<210>168
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>168
gaagtgtgat tggcaaaact ga 22
<210>169
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>169
gagctacgag ctgcctgac 19
<210>170
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>170
gtagtttcgt ggatgccaca 20
<210>171
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>171
ttagctgtgc tcgcgctact c 21
<210>172
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>172
tggttcacac ggcaggcata c 21
<210>173
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>173
agtgacaccc actcctcca 19
<210>174
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>174
tgtagccaaa ttcgttgtc 19
<210>175
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>175
ctcatggact gattatggac ag 22
<210>176
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>176
tccagcaggt cagcaaagaa 20
<210>177
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>177
taaaccctgc gtggcaatcc ct 22
<210>178
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>178
tgaacacaaa gcccacattc cc 22

Claims (7)

1. A PCR reagent for detecting angiogenesis signaling pathway gene expression, characterized in that the PCR reagent comprises the following primers:
(1) the PCR reaction primers for detecting the AKT1 gene have the following primer sequences:
5'- GGTGAAGGAGAAGGCCACA -3'；
5'- TACTTCAGGGCCGTCAGG -3'；
(2) the PCR reaction primer for detecting ANG gene has the following primer sequence:
5'- CTCCATGCCAGTACCGAG -3'；
5'- GACGACGGAAAATTGACTGA -3'；
(3) the PCR reaction primers for detecting the ANGPT1 gene have the following primer sequences:
5'- CAGTACAACACAAACGCTC -3'；
5'- CTCCGACTTCATGTTTTCCA -3'；
(4) the PCR reaction primers for detecting the ANGPT2 gene have the following primer sequences:
5'- ACTTTCGGAAGAGCATGGA -3'；
5'- GAGTCATCGTATTCGAGCG -3'；
(5) the PCR reaction primer for detecting the ANGPTL4 gene has the following primer sequence:
5'- TCCACCGACCTCCCGTT -3'；
5'- CCTCATGGTCTAGGTGCTTG -3'；
(6) the PCR reaction primer for detecting the ANPEP gene has the following primer sequence:
5'- TCAACATCACGCTTATCCACC -3'；
5'- GTCGAACTCACTGACAATGAAG -3'；
(7) the PCR reaction primer for detecting the BAI1 gene has the following primer sequence:
5'- CAACCTGGTTCTCAGCATCC -3'；
5'- GACGGTCGTGTTCCTCTG -3'；
(8) the PCR primers for detecting the CCL11 gene have the following primer sequences:
5'- CCCTTCAGCGACTAGAGAG -3'；
5'- CTTGGGGTCGGCACAGAT -3'；
(9) the PCR primers for detecting the CCL2 gene have the following primer sequences:
5'- CAGCCAGATGCAATCAATGC -3'；
5'- GGAATCCTGAACCCACTTCT -3'；
(10) the primer sequence of the PCR reaction primer for detecting the CDH5 gene is as follows:
5'- TTCACGCATCGGTTGTTCAA -3'；
5'- GCTTCCACCACGATCTCATA -3'；
(11) the PCR reaction primers for detecting the COL18A1 gene have the following primer sequences:
5'- GGTCTACGTGTCGGAGCA -3'；
5'- GCCTCGTTCGCCCTTAGA -3'；
(12) The PCR reaction primers for detecting the COL4A3 gene have the following primer sequences:
5'- CAGTGGTTCTAAGGGTGAGC -3'；
5'- AAAGCCAAAGAATCCCGGAG -3'；
(13) the PCR reaction primer for detecting the CTGF gene has the following primer sequence:
5'- AAAGTGCATCCGTACTCCCA -3'；
5'- CGTCGGTACATACTCCACAG -3'；
(14) PCR primers for detecting CXCL1 gene have the following primer sequences:
5'- TGTGAAGGCAGGGGAATGT -3'；
5'- GCCCCTTTGTTCTAAGCCA -3'；
(15) PCR primers for detecting CXCL10 gene have the following primer sequences:
5'- TGGCATTCAAGGAGTACCTC -3'；
5'- GATGGCCTTCGATTCTGGATT -3'；
(16) PCR primers for detecting CXCL5 gene have the following primer sequences:
5'- GCTGCGTTGCGTTTGTTTAC -3'；
5'- GGCGAACACTTGCAGATTAC -3'；
(17) PCR primers for detecting CXCL6 gene have the following primer sequences:
5'- GAGCTGCGTTGCACTTGTT -3'；
5'- CAGTTTACCAATCGTTTTGGGG -3'；
(18) PCR primers for detecting CXCL9 gene have the following primer sequences:
5'- CAGTAGTGAGAAAGGGTCGC -3'；
5'- GGGCTTGGGGCAAATTGTT -3'；
(19) the PCR reaction primers for detecting the EDN1 gene have the following primer sequences:
5'- AGGCAACAGACCGTGAAAAT -3'；
5'- GACCTGGTTTGTCTTAGGTG -3'；
(20) the PCR reaction primer for detecting the EFNA1 gene has the following primer sequence:
5'- CAGGCCCATGACAATCCAC -3'；
5'- TGACCGATGCTATGTAGAACC -3'；
(21) the PCR reaction primers for detecting the EFNB2 gene have the following primer sequences:
5'- TCAGCCCTAACCTCTGGGG -3'；
5'- CTCCAAAGACCCATTTGATGTA -3'；
(22) the PCR reaction primer for detecting EGF gene has the following primer sequence:
5'- CCTCACCCGATAATGGTGGA -3'；
5'- CAGGAAAGCAATCACATTCCC -3'；
(23) the PCR reaction primer for detecting the ENG gene has the following primer sequence:
5'- CATCCTTCGTGGAGCTACC -3'；
5'- AGGAGTGGTCTGGATCGG -3'；
(24) the PCR reaction primers for detecting the EPHB4 gene have the following primer sequences:
5'- TGTGAACCTGACTCGATTCC -3'；
5'- TCGGCACTTGGTGTTCCC -3'；
(25) the PCR reaction primers for detecting the ERBB2 gene have the following primer sequences:
5'- CTCATCGCTCACAACCAAGT -3'；
5'- ACAGGGGTGGTATTGTTCAG -3'；
(26) the PCR reaction primers for detecting the F3 gene have the following primer sequences:
5'- GAAGCAGACGTACTTGGCAC -3'；
5'- GGGAGTTCTCATACAGAGGC -3'；
(27) The PCR reaction primers for detecting the FGF1 gene have the following primer sequences:
5'- CACCGACGGGCTTTTATACG -3'；
5'- CCATTCTTCTTGAGGCCAAC -3'；
(28) the PCR reaction primers for detecting the FGF2 gene have the following primer sequences:
5'- GTGTGTGCTAACCGTTACCT -3'；
5'- CTGCCCAGTTCGTTTCAGTG -3'；
(29) the primer sequence of the PCR reaction primer for detecting the FGFR3 gene is as follows:
5'- CCAAGCCTGTCACCGTAG -3'；
5'- AGAAACTCCCGCAGGTTACC -3'；
(30) the PCR reaction primer for detecting the FIGF gene has the following primer sequence:
5'- CTCAGTGCAGCCCTAGAGAA -3'；
5'- AACACGTTCACACAAGGGG -3'；
(31) PCR reaction primers for detecting FLT1 gene have the following primer sequences:
5'- AAAACGCATAATCTGGGACAGT -3'；
5'- CGTGGTGTGCTTATTTGGA -3'；
(32) the primer sequence of the PCR reaction primer for detecting the FN1 gene is as follows:
5'- AGAATAAGCTGTACCATCGCAA -3'；
5'- GACCACATAGGAAGTCCCAG -3'；
(33) the PCR reaction primer for detecting HGF gene has the following primer sequence:
5'- CTATCGGGGTAAAGACCTACA -3'；
5'- GTAGCGTACCTCTGGATTGC -3'；
(34) the primer sequence of the PCR reaction primer for detecting the HIF1A gene is as follows:
5'- ACCACAGGACAGTACAGGAT -3'；
5'- CGTGCTGAATAATACCACTCAC -3'；
(35) the PCR reaction primer for detecting the HPSE gene has the following primer sequence:
5'- AAGGTGTTAATGGCAAGCGT -3'；
5'- AAAGGATAGGGTAACCGCAAG -3'；
(36) the PCR reaction primers for detecting the ID1 gene have the following primer sequences:
5'- TGCTCTACGACATGAACGG -3'；
5'- AAGGTCCCTGATGTAGTCGAT -3'；
(37) the PCR reaction primers for detecting the IFNA1 gene have the following primer sequences:
5'- CCTCGCCCTTTGCTTTACT -3'；
5'- CTGTGGGTCTCAGGGAGATC -3'；
(38) the PCR reaction primer for detecting the IFNG gene has the following primer sequence:
5'- CGGTAACTGACTTGAATGTCCA -3'；
5'- CGCTTCCCTGTTTTAGCTGC -3'；
(39) the PCR reaction primer for detecting IGF1 gene has the following primer sequence:
5'- AAGATGCTCACCATGTC -3'；
5'- AGGGGCTTTTATTTCAACA -3'；
(40) the PCR reaction primers for detecting the IL1B gene have the following primer sequences:
5'- TCGACACATGGGATAACGAGG -3'；
5'- TTTTTGCTGTGAGTCCCGGAG -3'；
(41) the primer sequence of the PCR reaction primer for detecting the IL6 gene is as follows:
5'- CTGAACCTTCCAAAGATGGC -3'；
5'- TCACCAGGCAAGTCTCCTCA -3'；
(42) The primer sequence of the PCR reaction primer for detecting the IL8 gene is as follows:
5'- TTTGCCAAGGAGTGCTAAAGA -3'；
5'- ACCCTCTGCACCCAGTTTTC -3'；
(43) the PCR reaction primers for detecting the ITGAV gene have the following primer sequences:
5'- CCCATTGTACCATTGGAGAAC -3'；
5'- GGAGCATACTCAACAGTCTTTG -3'；
(44) the primer sequence of the PCR reaction primer for detecting the ITGB3 gene is as follows:
5'- GTGACCTGAAGGAGAATCTGC -3'；
5'- CGGAGTGCAATCCTCTGG -3'；
(45) the primer sequence of the PCR reaction primer for detecting the JAG1 gene is as follows:
5'- CTCCAGCCGGTGAAGACA -3'；
5'- CCTGGTGACATCATCTCCTTGT -3'；
(46) the PCR reaction primers for detecting the KDR gene have the following primer sequences:
5'- ACGTGTCACTTTGTGCAAGA -3'；
5'- TCCATGAGACGGACTCAGAA -3'；
(47) the PCR reaction primers for detecting the LECT1 gene have the following primer sequences:
5'- CATGACATTCGACCCTAGAC -3'；
5'- CAGCTACATGGCATGATGAC -3'；
(48) the PCR reaction primers for detecting the LEP gene have the following primer sequences:
5'- GCCTTCCAGAAACGTGATCC -3'；
5'- TCTGTGGAGTAGCCTGAAGC -3'；
(49) the PCR reaction primer for detecting the MDK gene has the following primer sequence:
5'- GCGGTCGCCAAAAAGAAAG -3'；
5'- ACTTGCAGTCGGCTCCAAAC -3'；
(50) the PCR reaction primer for detecting MMP14 gene has the following primer sequence:
5'- ATCTGTGACGGGAACTTTGA -3'；
5'- GCAGTGTTGATGGACGCA -3'；
(51) the PCR reaction primer for detecting MMP2 gene has the following primer sequence:
5'- ATACCCCTTTGACGGTAAGGA -3'；
5'- CCTTCTCCCAAGGTCCATAG -3'；
(52) the PCR reaction primer for detecting MMP9 gene has the following primer sequence:
5'- GACGGGTATCCCTTCGACG -3'；
5'- AAACCGAGTTGGAACCACGA -3'；
(53) the primer sequence of the PCR reaction primer for detecting the NOS3 gene is as follows:
5'- CGCACTTCTGTGCCTTTGCTC -3'；
5'- GCTCGGGTGGATTTGCTGCTC -3'；
(54) the primer sequence of the PCR reaction primer for detecting the NOTCH4 gene is as follows:
5'- TAGGGGCTCTTCTCGTCCT -3'；
5'- AACTTCTGCCTTTGGCTTC -3'；
(55) the PCR reaction primers for detecting the NRP1 gene have the following primer sequences:
5'- CGTGGAAGTCTTCGATGGAG -3'；
5'- ACCATGTGTTTCGTAGTCAGA -3'；
(56) the PCR reaction primers for detecting the NRP2 gene have the following primer sequences:
5'- CAACGGGACCATCGAATCTC -3'；
5'- CAGCCAATCGTACTTGCAGT -3'；
(57) The PCR reaction primers for detecting the PDGFA gene have the following primer sequences:
5'- GCAAGACCAGGACGGTCATT -3'；
5'- GCACTTGACACTGCTCGT -3'；
(58) the PCR reaction primers for detecting the PECAM1 gene have the following primer sequences:
5'- CCGTGACGGAATCCTTCTCT -3'；
5'- CTGGACTCCACTTTGCAC -3'；
(59) the PCR reaction primers for detecting the PF4 gene have the following primer sequences:
5'- TTAACGGAGAGCCTGCTGAG -3'；
5'- GCCAGTTGCAAATGGCATTAGAAGGG -3'；
(60) the PCR reaction primers for detecting the PGF gene have the following primer sequences:
5'- AACGGCTCGTCAGAGGTG -3'；
5'- CAGTGCAGATTCTCATCGCC -3'；
(61) the PCR reaction primers for detecting the PLAU gene have the following primer sequences:
5'- CTTGTCCAAGAGTGCATGGT -3'；
5'- CAGGGCTGGTTCTCGATGG -3'；
(62) the PCR reaction primers for detecting the PLG gene have the following primer sequences:
5'- GCGACATTCTTGAGTGTGAAG -3'；
5'- CGGGGTTACGACAGTAATTCTT -3'；
(63) the PCR reaction primers for detecting the PROK2 gene have the following primer sequences:
5'- TGACAAGGACTCCCAATGTG -3'；
5'- GACCCAGATACTGACAGCAC -3'；
(64) the PCR reaction primers for detecting the PTGS1 gene have the following primer sequences:
5'- TCTGTGCCTAAAGATTGCCC -3'；
5'- TCTCCATAAATGTGGCCGAG -3'；
(65) the PCR reaction primers for detecting the S1PR1 gene have the following primer sequences:
5'- CTGCTGGCAAATTCAAGCGA -3'；
5'- TTGTCCCCTTCGTCTTTCTG -3'；
(66) the PCR reaction primer for detecting the SERPINE1 gene has the following primer sequence:
5'- GTGGACTTTTCAGAGGTGGA -3'；
5'- GCCGTTGAAGTAGAGGGCATT -3'；
(67) the PCR reaction primers for detecting the SERPINF1 gene have the following primer sequences:
5'- GAAATTCCCGATGAGATCAGC -3'；
5'- GTCAAACTTTGTTACCCACTGC -3'；
(68) the PCR reaction primers for detecting the SPHK1 gene have the following primer sequences:
5'- GAGTGGGTTCCAAGACACCT -3'；
5'- GGTGCAGCAAACATCTCAC -3'；
(69) the PCR reaction primers for detecting the TEK gene have the following primer sequences:
5'- AGGATACGAACCATGAAGATGC -3'；
5'- GGGCACTGAATGGATGAAG -3'；
(70) the PCR reaction primers for detecting the TGFA gene have the following primer sequences:
5'- GGTCCGAAAACACTGTGAGT -3'；
5'- GCAAGCGGTTCTTCCCTTC -3'；
(71) the PCR reaction primers for detecting the TGFB1 gene have the following primer sequences:
5'- AATTCCTGGCGATACCTCAG -3'；
5'- CACAACTCCGGTGACATCAA -3'；
(72) The PCR reaction primers for detecting the TGFB2 gene have the following primer sequences:
5'- CATCCCGCCCACTTTCTAC -3'；
5'- GCTCAATCCGTTGTTCAGGC -3'；
(73) the PCR reaction primers for detecting the TGFBR1 gene have the following primer sequences:
5'- CGGCGTTACAGTGTTTCTG -3'；
5'- GCACATACAAACGGCCTATCT -3'；
(74) the PCR reaction primers for detecting the THBS1 gene have the following primer sequences:
5'- AGACTCCGCATCGCAAAG -3'；
5'- CACCACGTTGTTGTCAAGGG -3'；
(75) the PCR reaction primers for detecting the THBS2 gene have the following primer sequences:
5'- CCCTCCTAAGACAAGGAACA -3'；
5'- CACCCACGTTTCATTTTCCG -3'；
(76) the primer sequence of the PCR reaction primer for detecting the TIE1 gene is as follows:
5'- ATTGCCGACTCCAGTGCC -3'；
5'- TCTGGCTTGCGTAGCTCT -3'；
(77) the primer sequence of the PCR reaction primer for detecting the TIMP1 gene is as follows:
5'- CCACCTTATACCAGCGTTATGA -3'；
5'- GTGTAGACGAACCGGATGTC -3'；
(78) the primer sequence of the PCR reaction primer for detecting the TIMP2 gene is as follows:
5'- CTGCGAGTGCAAGATCAC -3'；
5'- GGTGCCCGTTGATGTTCTTC -3'；
(79) the primer sequence of the PCR reaction primer for detecting the TIMP3 gene is as follows:
5'- TGCAACTTCGTGGAGAGGTG -3'；
5'- AGGTGATACCGATAGTTCAGC -3'；
(80) the PCR reaction primer for detecting TNF gene has the following primer sequence:
5'- CTCTCTCTAATCAGCCCTCTG -3'；
5'- GAGGACCTGGGAGTAGATGAG -3'；
(81) the PCR reaction primer for detecting the TYMP gene has the following primer sequence:
5'- GTGTGGGTGACAAGGTCAG -3'；
5'- CAGCACTTGCATCTGCTC -3'；
(82) the PCR reaction primers for detecting the VEGFA gene have the following primer sequences:
5'- CAACATCACCATGCAGATTATGC -3'；
5'- TCGGCTTGTCACATTTTTCTTG -3'；
(83) the PCR reaction primers for detecting the VEGFB gene have the following primer sequences:
5'- AGATGTCCCTGGAAGAACACA -3'；
5'- AGTGGGATGGGTGATGTCAG -3'；
(84) the PCR reaction primers for detecting the VEGFC gene have the following primer sequences:
5'- TGTGTGTCCGTCTACAGATGT -3'；
5'- GAAGTGTGATTGGCAAAACTGA -3'；
(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:
5'- GAGCTACGAGCTGCCTGAC -3'；
5'- GTAGTTTCGTGGATGCCACA -3'；
(86) the PCR reaction primers for detecting the B2M gene have the following primer sequences:
5'- TTAGCTGTGCTCGCGCTACTC -3'；
5'- TGGTTCACACGGCAGGCATAC -3'；
(87) The PCR reaction primers for detecting the GAPDH gene have the following primer sequences:
5'- AGTGACACCCACTCCTCCA -3'；
5'- TGTAGCCAAATTCGTTGTC -3'；
(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:
5'- CTCATGGACTGATTATGGACAG -3'；
5'- TCCAGCAGGTCAGCAAAGAA -3'；
(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:
5'- TAAACCCTGCGTGGCAATCCCT -3'；
5'- TGAACACAAAGCCCACATTCCC -3'。
2. use of the PCR reagent of claim 1 in the preparation of a detection reagent for detecting tumor cell angiogenesis signaling pathway gene expression.
3. The use of the PCR reagent according to claim 2 for preparing a detection reagent for detecting tumor cell angiogenesis signaling pathway gene expression, characterized in that: the tumor cells are liver cancer cells, breast cancer cells, lung cancer cells and stomach cancer cells.
4. Use of a PCR reagent according to claim 2 for the preparation of a detection reagent for the detection of angiogenesis signaling pathways in tumor cells, characterized in that: the detection reagent is used for detecting by using a real-time fluorescent quantitative PCR detection method.
5. The use of the PCR reagent according to claim 4 for preparing a detection reagent for detecting angiogenesis signaling pathway of tumor cells, wherein: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1-0.2 mul of upstream primer, 0.1-0.2 mul of downstream primer, 25-100ng of cDNA template, and sterile distilled water to make up to 20 mul.
6. Use of a PCR reagent according to claim 5 in the preparation of a detection reagent for detecting angiogenesis signaling pathways in tumor cells, wherein: the Taq polymerase is 2X SYBR Premix Ex Taq polymerase of TaKaRa company.
7. The use of the PCR reagent according to claim 4 for preparing a detection reagent for detecting angiogenesis signaling pathway of tumor cells, wherein: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 deg.C for 5 seconds, 59 deg.C for 20 seconds, and 40 cycles; the dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
CN202010666819.7A 2020-07-13 2020-07-13 PCR reagent for detecting gene expression of cell angiogenesis signal pathway and application thereof Pending CN111850123A (en)

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