CN103751805B - Interference SIRT1 expresses the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal - Google Patents
Interference SIRT1 expresses the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal Download PDFInfo
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Abstract
The invention belongs to chemical field, disclose interference SIRT1 and express the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal, express multiplication capacity, clonality and the balling-up ability that significantly can reduce liver-cancer stem cell by suppressing SIRT1 in liver-cancer stem cell, thus suppress the self renewal of liver-cancer stem cell; Therefore interference SIRT1 can be expressed the self renewal of reagent targeted inhibition liver-cancer stem cell, realize the targeted therapy of hepatocarcinoma.
Description
Technical field
The invention belongs to chemical field, particularly disturb SIRT1 to express the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal.
Background technology
Tumor is the disease of caused a class complexity of being morphed by hereditary change or epigenetic, and tumor occurs and evolution is a complicated multi-step process.Recent study finds, there is a small set of tumor cell subgroup with stem-like cell in tumor, there is the ability opening beginning tumor, be therefore called as tumor stem cell (Cancer Stem Cells, CSCs) or tumor open beginning cell (Tumor Initiating Cells, TICs).Up to now, from least 30 kinds of kinds of tumor such as hepatocarcinoma, breast carcinoma, colorectal cancer, glioma, pulmonary carcinoma, carcinoma of prostate, melanoma and leukemia, separation andpreconcentration goes out tumor stem cell.Therefore, CSCs molecular regulation mechanism and there is in malignant tumor the key scientific problems that the developing mechanism of action has become tumor research field.
In recent years, epigenetics modifies the hot subject increasingly becoming tumor research.Epigenetic modification is as important binding mode a kind of in molecular regulation mechanism; take part in a series of important biological processes such as cell self-renewal, propagation and differentiation, and wherein Mechanisms of Histone Acetylation Modification as a kind of important posttranscriptional modification of epigenetic modification.Acetylation modification and the system of gene expression in eukaryote of histone are closely related, are a kind of important mechanism of gene transcription regulation.Existing research also shows, the acetylation modification of histone plays a significant role in embryonic stem cell self renewal and atomization, but have not been reported in liver-cancer stem cell research.
SIRT1 albumen is a kind of nicotinamide adenine dinucleotide (NAD
+) dependent Class III histone deacetylases, belong to induction reticent adjustment 2(silent information regulator2, Sir2) family.SIRT1 has important role in the generation of tumor and the metabolism of cell, and its effect extensively, comprises cell ageing, and DNA repairs, cell cycle, metabolism, the generation etc. of stress and cancer.Mainly concentrate in the embryonic stem cell of Mus and people and the hematopoietic stem cell of people to SIRT1 in the research of stem cell at present, but SIRT1 is at adjustment entity tumor especially in the expression of liver-cancer stem cell, and its effect of playing and molecular mechanism have not yet to see report.
Summary of the invention
In view of this, interference SIRT1 is the object of the present invention is to provide to express the novelty teabag of reagent, for the targeted therapy of hepatocarcinoma is significant.
For achieving the above object, the invention provides following technical scheme:
Interference SIRT1 expresses the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal.
Preferably, described interference SIRT1 expresses reagent is the slow virus that interference SIRT1 expresses.
Preferred, the slow virus that described interference SIRT1 expresses contains Lv-sh-SIRT1 plasmid.
Preferred, the slow virus disturbing SIRT1 to express described in the slow virus that described interference SIRT1 expresses by after Lv-sh-SIRT1 plasmid transfection 293T cell, through packing, purification and obtaining.
Preferably, SIRT1 is disturbed to express the application of reagent in the medicine of preparation suppression liver-cancer stem cell propagation.
Preferably, SIRT1 is disturbed to express the application of reagent in the medicine of preparation suppression liver-cancer stem cell Clone formation.
Preferably, SIRT1 is disturbed to express the application of reagent in the medicine of preparation suppression liver-cancer stem cell balling-up.
Beneficial effect of the present invention is: the invention discloses interference SIRT1 and express the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal, make use of propagation, Clone formation, balling-up ability and the subcutaneous transplantation neoplasia rate that can suppress liver-cancer stem cell by suppressing SIRT1 to express in liver-cancer stem cell, delay tumor growth in vivo, therefore interference SIRT1 can be expressed reagent and be used for targeted therapy of liver cancer, significant to prolongation hepatocarcinoma life span.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is for detecting at Nanog
posliver-cancer stem cell and Nanog
negnon-liver cancer stem cell in SIRT1 expression (A: by western-blot detect SIRT1 express; B: the expression being detected SIRT1 by immunofluorescence dyeing).
Fig. 2 be detect in immunodeficient mouse subcutaneous transplantation tumor SIRT1 express (A:western-blot detect express at SIRT1; B:H & E section and immunohistochemical staining detect the expression of SIRT1).
Fig. 3 is the expression detecting SIRT1 in the liver-cancer stem cell atomization of NanogPos.
Fig. 4 is the jamming effectiveness that western-blot checking knocks out SIRT1.
Fig. 5 is that after the expression of interference SIRT1, CCK-8 detects cell proliferation.
Fig. 6 detects clonality after the expression of interference SIRT1.
Fig. 7 detects balling-up ability after the expression of interference SIRT1.
Fig. 8 is weight and volume (the A:NOD/SCID mouse subcutaneous transplanting tumor picture of NOD/SCID mouse subcutaneous transplanting tumor; The tumor weight of B:NOD/SCID mouse subcutaneous transplanting tumor; The gross tumor volume of C:NOD/SCID mouse subcutaneous transplanting tumor).
Fig. 9 is that SABC detects the expression of SIRT1 or Ki-67 in transplanted tumor.
Figure 10 is that different cell number becomes tumor ability (A: the Retention of different cell number in NOD/SCID Mus subcutaneous transplantation tumor; B: stem cell forms ratio).
Figure 11 is the hepatocyte in situ injection detection NOD/SCID mouse survival time.
Figure 12 is that western-blot checking is at Nanog
negprocess LAN SIRT1 result figure.
Figure 13 detects Clone formation and balling-up ability (A: Clone formation after process LAN SIRT1; B balling-up ability).
Figure 14 be detected the NOD/SCID mice-transplanted tumor of expressing SIRT1 tumor size and weight (A: transplanted tumor become tumor situation; B: the weight of transplanted tumor; C: the volume of transplanted tumor).
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the such as Molecular Cloning: A Laboratory guide (third edition, J. Pehanorm Brooker etc. work) described in condition, Shan et al.2012, the condition that conditioned disjunction described in hepatology, 56:1014-1014 is advised according to manufacturer.
The liver-cancer stem cell model that the present invention uses is built by this laboratory, this model contains the Lentiviral (Nanog/GFP reporting system) that Nanog promoter regulation GFP reporter gene builds, its construction method see Shan et al.2012, hepatology, 56:1014-1014; Lv-sh-Scramble plasmid and Lv-sh-SIRT1 plasmid, adenovirus vector AD-GFP are built by this laboratory, specifically see Shan et al.2012, and hepatology, 56:1014-1014; HEK293 cell is preserved by this laboratory.Adenovirus AD-SIRT1 is so kind as to give by Zhu Weiguo professor, and its construction method is shown in Liu et al.2011, and Proc NatlAcad Sci U S A.108:1925-1930.
Human primary liver cancer cell line PLC/PRF/5 and hepatoma cell line Huh7 is purchased from Shanghai cell institute of the Chinese Academy of Sciences; Anti-SIRT1 antibody purchased from American Santa Cruz company, anti-Albumin antibody purchased from American Abcam company.Envision
tMwith DAB nitrite ion purchased from DAKO company of Denmark.
One, the expression of SIRT1 in liver-cancer stem cell and non-liver cancer stem cell is detected
Get human primary liver cancer cell line PLC/PRF/5, hepatoma cell line Huh7 and hepatoma carcinoma cell T1224, (be designated as Nanog with flow cytometer difference sorting Nanog masculine liver cancer stem cell
pos) and the negative non-liver cancer stem cell of Nanog (be designated as Nanog
neg), then western-blot method detects the expression of SIRT1, is specially: collect 1 × 10 respectively
6cell number, centrifugal segregation PBS, adds protein lysate, acts on 60min on ice, drip 1 × sds gel sample loading buffer respectively, boil 10min at 100 DEG C of metal baths ,-20 DEG C of preservations, prepare 10% separation gel and 5% concentrated glue carries out electrophoresis, then transferring film and development, its result as shown in Figure 1A.From Figure 1A, Nanog
posthree kinds of liver-cancer stem cells in the expression of SIRT1 significantly increase.
By the Nanog of separation three kinds of cell lines
posand Nanog
negdetected the expression of SIRT1 by immunofluorescence dyeing, be specially: by the Nanog of separation three kinds of cell lines
posand Nanog
negcentrifugal segregation PBS respectively, be planted in respectively on creep plate, culture fluid is the DMEM (v/v) containing 10%FBS, treat that cell grows to creep plate and is about 50-60%, discard culture fluid, PBS quick wash 3 times, then be that 4% paraformaldehyde fixative is fixed with mass fraction, room temperature fixes 30min, PBS solution rinsing 3 times, each 5min; Then PBS solution is discarded, drip the TritonX-100 solution that mass fraction is 0.1%, room temperature effect 5min, then use PBS solution rinsing 3 times, each 5min, PBS solution, drip the PBS solution (v/v) containing 10%FBS, room temperature closes 30min, discards confining liquid, drip 1:300(volume ratio) the anti-SIRT1 antibody of rabbit that dilutes, 4 DEG C of overnight incubation; With PBS rinsing 3 times, each 5min, then add 1:1000(volume ratio) rabbit anti goat igg (purchased from American Invitrogen company) the incubated at room 60min of 488 labellings that dilutes; Then use PBS rinsing 3 times, each 5min minute, then drip 1:1000(volume ratio) the DAPI solution that dilutes, room temperature effect 10min; After PBS rinsing, creep plate is inverted on microscope slide, with the PBS mounting (v/v) containing 40% glycerol.Confocal laser scanning microscope coloration result, result as shown in Figure 1B.From Figure 1B, at Nanog
posthree kinds of liver-cancer stem cells in the expression of SIRT1 be significantly higher than Nanog
negthree kinds of non-liver cancer stem cell.
Two, SIRT1 is at the expression of liver-cancer stem cell and non-liver cancer stem cell transplantation struma
For studying the expression of SIRT1 liver-cancer stem cell and non-liver cancer stem cell further, Nanog masculine liver cancer stem cell and the negative non-liver cancer stem cell of Nanog of the sorting of subcutaneous injection PLC/PRF/5 cell line is carried out in NOD/SCID Mus body, specific as follows: to utilize flow cytometer sorting Nanog masculine liver cancer stem cell and the negative non-liver cancer stem cell of Nanog in PLC/PRF/5 cell, centrifugal segregation PBS, be planted in respectively in 6 orifice plates and continue to cultivate to expand cell quantity, culture fluid is the DMEM(v/v containing 10%FBS); Then 1.2 × 10 are got respectively
7the negative non-liver cancer stem cell of individual Nanog masculine liver cancer stem cell and Nanog, centrifugal segregation culture fluid, it is resuspended to add 1.2mL PBS, does transplanted tumor experiment, every only injection 1 × 10 under being then expelled to NOD/SCID Corium Mus respectively
6cell number, point 10 some injections, after surrounding, put to death mice, peel off tumor, portion of tissue is for extracting tumor tissues albumen, and identify the expression of SIRT1 with western-blot, result is as shown in Figure 2 A; Remaining tissue is used for paraffin embedding and cuts into slices, and identifies the expression of SIRT1 after immunohistochemical staining.Concrete grammar is: by fixing NOD/SCID Mus subcutaneous transplantation tumor, dewater and use paraffin embedding, H & E dyes and serial section, often open 4-5 μm thick, sheet is mounted with SABC slide, room temperature is placed for subsequent use, then by the roasting sheet instrument baking section 20min of section, put into dimethylbenzene, dewaxing 10-20min, be 70%-100% graded ethanol aquation by volume fraction again, distilled water rinsing is cut into slices 3 times, each 5min, then section to be placed in by 41mL citric acid and 9mL sodium citrate and the pH of standardize solution most 500mL is the liquor sodii citratis of 6.0, under 800W condition after Microwave method 3.30min to solution boiling, under 150W condition, 16min maintains fluidized state again, to cold water, room temperature is cooled to after reparation, distilled water rinsing is cut into slices 3 times, each 5min, then the H of mass fraction 3% is dripped by 50 μ L/ sheets
2o
2solution, hatches 20min for 37 DEG C, after distilled water rinsing 5min, then cuts into slices 2 times with PBS rinsing, each 5min, with dripping 1:300(v/v) the anti-SIRT1 antibody of rabbit that dilutes, 4 DEG C of overnight incubation, taking-up next day PBS rinsing is cut into slices 3 times, each 5min, then drips EnVision by 50 μ L/ sheets
tMtwo resist, and hatch 30min for 37 DEG C, PBS rinsing is cut into slices 3 times, each 5min, drips DAB nitrite ion after rinsing, tap water cessation reaction, then with haematoxylin lining dye 45s, hydrochloride alcohol breaks up, and tap water, oil blackeite, finally use volume fraction 70%-100% gradient alcohol dehydration, dimethylbenzene is transparent, resinene mounting, microscopic examination, result as shown in Figure 2 B.
Immunohistochemical staining carries out H & E Coloration experiment simultaneously, is specially, get transplanted tumor conveniently pathological tissue fix, dewater, paraffin embedding, and serial section, slice thickness 3-5 μm; To mount after sheet with roasting sheet instrument baking section 20min, put into dimethylbenzene and dewax after 10-20min, be 100%-75% gradient alcohol dehydration by volume fraction, then cut into slices 3 times with distilled water rinsing, each 5min; Again section is put into haematoxylin liquid contaminate section 5min, then in clear water rinsing haematoxylin liquid; Then with mass fraction be 1% hydrochloride alcohol differentiation, running water, basis of microscopic observation Color; Again section is placed in eosin stain to dye 30s, by 75%-100% graded ethanol aquation; Dry ethanol in last baking oven, immerse humidifying section in dimethylbenzene, section observed by resinene solution mounting, and result as shown in Figure 2 B.
Result shows, with Nanog
negnon-liver cancer stem cell transplantation tumor compare, at Nanog
postransplanted tumor in the expression of SIRT1 significantly raise, immunohistochemical staining result also obtains identical result.More than research shows, in liver-cancer stem cell, the expression of SIRT1 is significantly higher than non-liver cancer stem cell, shows that high expressed and liver-cancer stem cell exist internal relation further.
Three, the expression of SIRT1 in liver-cancer stem cell atomization
For studying the change of SIRT1 expression in liver-cancer stem cell atomization further, by human primary liver cancer cell line PLC/PRF/5, hepatoma cell line Huh7 by selected by flow cytometry apoptosis Nanog
posliver-cancer stem cell carry out In vitro culture, collect 0 day respectively, 4 days, 8 days, 12 days, 16 days, 20 days, the albumen of 24 days different time points, utilize western-blot to detect the expression of SIRT1, result is as shown in Figure 3.Result is presented in liver-cancer stem cell atomization, and the expression of SIRT1 is lowered gradually along with the prolongation of divergaence time, show the overexpression of SIRT1 in liver-cancer stem cell, and its expression is lowered gradually in the atomization of liver-cancer stem cell.
Four, at Nanog
posliver-cancer stem cell in lower the impact of expression on liver-cancer stem cell of SIRT1
1. the packaging of slow virus Lv-sh-SIRT1 and Lv-sh-Scramble
At the culture dish middle berth 2 ~ 5 × 10 of 10cm
6individual 293T cell (preferably before 20 generations, the state of cell is required good, the speed of growth is very fast, otherwise it is very large on viral yield impact), culture fluid is containing the DMEM(v/v of 10%FBS), when cell confluency to 70%, renew the fresh DMEM(v/v containing 10%FBS) culture fluid, for transfection after 3-4h.
Get Lv-sh-SIRT1 or the Lv-sh-Scramble slow virus expression plasmid of 15-20 μ g, drip the CaCl of 50 μ L2.5M respectively
2mixing, sterilized water is added respectively to cumulative volume 450 μ L in mixed liquor, 500 μ L2 × HBS buffer (2 × HBS buffer shakes before using on turbula shaker) are added after mixing, then be placed on turbula shaker and shake more than 10s, drip after terminating, continue concussion, after room temperature places 5 minutes, liquid is added in the 293T of cultivation, after adding, rocks mixing all around, add the DMEM(v/v that rear 6-12 hour renews fresh 10%FBS) culture fluid.
2. the collection of slow virus Lv-sh-SIRT1 and Lv-sh-Scramble and purification
Start respectively to collect a cell culture fluid in latter 48 hours, 72 hours from transfection, after 48 hr collections, supplement the culture fluid of 15mL again, the virus of collecting centrifugal 10min under 1000rpm condition is removed cell debris, collect supernatant, and with the membrane filtration of 0.45 μm, filtrate is transferred to the evaporating column of Millopore, is concentrated into 1mL(to the liquid volume in the groove of upper strata and can adds several times 5000rpm is centrifugal).By concentrated solution subpackage, be placed in-80 DEG C of cryogenic refrigerators and preserve.
3. at Nanog
posliver-cancer stem cell infect Lv-sh-SIRT1 or Lv-sh-Scramble slow virus
Get human primary liver cancer cell line PLC/PRF/5, hepatoma cell line Huh7 and hepatoma carcinoma cell T1224, by selected by flow cytometry apoptosis Nanog
posliver-cancer stem cell, be then seeded in 6 orifice plates, at 37 DEG C, 5%CO
2incubator is cultivated, and treats that cell grows to 5 × 10
5time, liquid in sucking-off hole, adds the 10%FBS/DMEM(v/v of 1mL) culture fluid, then every hole is with 10 MOI values, and add Lv-sh-SIRT1 or Lv-sh-Scramble slow virus, blank group does not add slow virus, then in 37 DEG C, 5%CO
2incubator is cultivated, and after 6-8 hour, liquid in sucking-off hole, adds the 10%FBS/DMEM culture fluid of 2mL, after 72h, and the effect that the expression detecting downward SIRT1 is respectively formed liver-cancer stem cell propagation, Clone formation, balling-up.
First, utilize western-blot to detect the protein expression level of interference SIRT1 virus 72h, the effectiveness of checking interference SIRT1, as hereinbefore, testing result as shown in Figure 4 for its detection method.Result shows, relative to the blank group of mock() and transfection Lv-sh-Scramble slow virus group (matched group), in three kinds of liver-cancer stem cells of transfection Lv-shSIRT1 slow virus, SIRT1 protein level is lowered all significantly, and it is effective for showing that interference SIRT1 expresses.
Then, utilize CCK-8 method to detect the multiplication rate of latter 1st day, the 2nd day, the 3rd day, the 4th day, the 5th day of interference SIRT1 expression and the 6th day three kinds of liver-cancer stem cells, and detect absorbance at 450nm place, result as shown in Figure 5.Result shows, and through the detection of continuous 6 days, transfection Lv-sh-SIRT1 slow virus liver-cancer stem cell multiplication rate, significantly lower than matched group and blank group, showed to disturb the expression of SIRT1 significantly can suppress the multiplication capacity of liver-cancer stem cell.Simultaneously, three kinds of liver-cancer stem cells after infection Lv-sh-SIRT1 or Lv-sh-Scramble slow virus 72h are counted 200 respectively and is inoculated in 24 orifice plates (3 repetitions are set), cultivate and observe clonality after 14 days, count Clone formation number respectively, result as shown in Figure 6.Result shows, the three kinds of liver-cancer stem cell cells large peanut of formed clone (11.3 ± 1.5%) of transfection Lv-sh-SIRT1 slow virus are significantly less than compared with control cells (32.2 ± 0.6%), and both significant difference (PLC/PRF/5:P<0.0002; Huh7:P<0.0012; T1224:P<0.0218), show that the expression of lowering SIRT1 can reduce liver-cancer stem cell clonality.
For studying the expression of downward SIRT1 further to the impact of liver-cancer stem cell balling-up ability, three kinds of liver-cancer stem cells after infection Lv-sh-SIRT1 or Lv-sh-Scramble slow virus 72h are counted 10 cells and are inoculated in 96 orifice plates by respectively, 8 repetitions are set, cultivate and within 12 days, observe liver-cancer stem cell balling-up ability.By microscopic examination and counting, result as shown in Figure 7.Found that, in liver-cancer stem cell, lower the balling-up number (average is 10.2 ± 1.5%) that SIRT1 expresses becomes sphere volume little and number is few (average is 29.0 ± 0.7%) compared with cellular control unit, and both significant difference (PLC/PRF/5:P<0.0003; Huh7:P<0.0212; T1224:P<0.0349), illustrate lower that SIRT1 expresses can the balling-up energy for growth of T suppression cell.Above result of study shows that the expression of downward SIRT1 significantly reduces the self-renewal capacity of liver-cancer stem cell.
4. at Nanog
posin liver-cancer stem cell, lower the expression of SIRT1 to the impact of liver-cancer stem cell subcutaneous transplantation tumor energy for growth
Utilize the PLC/PRF/5 cell line Nanog be separated
poscell infects 1 × 10 respectively
6lv-sh-SIRT1 and the Lv-sh-Scramble slow virus of quantity, is then inoculated in that immunodeficiency NOD/SCID is subcutaneous sets up subcutaneous transplantation tumor model, with pentobarbital sodium intraperitoneal anesthesia NOD/SCID Mus during injection, then carries out subcutaneous injection.After inoculation slow virus cell 2 weeks, within every 2 days, observe the tumor growth state of NOD/SCID Mus, record mouse growth situation, tumor formation rate, measurement tumor growth situation (size).Process mice successively after inoculation slow virus cell 4-9 week, take a picture, collect transplanted tumor, and measure gross tumor volume and weight, a part is preserved liquid nitrogen and is filled with, and a part extracts histone, and residue tissue is fixed with 4% paraformaldehyde, and carries out specimens paraffin embedding slices.
During inoculation slow virus cell 4 weeks, de-cervical approach puts to death mice, and be separated tumor, then observe, its result as shown in Figure 8 A.Result shows, and compare with blank group with matched group, the mouse tumor volume of transfection Lv-shSIRT1 slow virus obviously reduces.Then the Nanog of inoculation slow virus is added up
poscell is gross tumor volume and weight in 26 days after inoculation, and draw time point transplanted tumor volume and tumor growth curve according to result, and result as shown in figs. 8 b and 8 c.After this result demonstrates transfection Lv-shSIRT1 slow virus equally, in NOD/SCID Mice Body, form tumor growth rate and volume obviously reduces.The above results indicates interference SIRT1 and expresses self-renewal capacity and the tumor growth that can suppress liver-cancer stem cell.
Get the inoculation slow virus cell tumor tissues of 4 weeks, carry out the expression of H & E dyeing and SABC detection SIRT1 and ki67 respectively, result as shown in Figure 9.As seen from the figure, matched group high expressed SIRT1 and Ki67, expresses SIRT1 and Ki67 expression after interference SIRT1 expresses and significantly reduces.
According to method same as described above, inoculate 1 × 10 respectively subcutaneous often of NOD/SCID mice
2, 1 × 10
3with 1 × 10
4the infection Lv-sh-SIRT1 of varying number level and the Nanog of Lv-sh-Scramble slow virus
poscell, adds up into tumor number after inoculating 9 weeks, then utilize online software, calculates the cell proportion wherein with tumor, shown in its result Figure 10.Result shows, and after interference SIRT1 expresses, each subcutaneous transplantation tumor needs 16927 liver-cancer stem cells; And matched group only needs 1350 liver-cancer stem cells, there is significant difference (p=4.52e) between the two.
For confirming to disturb SIRT1 to express self-renewal capacity and the tumor growth that can suppress liver-cancer stem cell further, utilize the PLC/PRF/5 cell line Nanog be separated
poscell infects 1 × 10 respectively
6lv-sh-SIRT1 and the Lv-sh-Scramble slow virus of quantity, is then inoculated in immunodeficiency NOD/SCID liver and sets up hepatocarcinoma Orthotopic Transplantation Model respectively.After inoculation slow virus cell, detect the impact on the NOD/SCID mouse survival time, result as shown in figure 11.As shown in Figure 11, compared with matched group, after interference SIRT1, obviously can increase the time-to-live of NOD/SCID mice.Above experimental result prompting, the expression of interference SIRT1 obviously inhibits the one-tenth tumor ability in liver-cancer stem cell body and extends the life span of NOD/SCID mice.
Five, at Nanog
negnon-liver cancer stem cell in raise the expression of SIRT1 to the impact of non-liver cancer stem cell
1.Ad-SIRT1 and AD-GFP adenovirus amplifies
With the T25Flask culture bottle paving HEK293 cell of 5mL, culture fluid is the DMEM containing 10%FBS, then at 37 DEG C, 5%CO
2incubator is cultivated, by the time, during cell confluency to 70%, change culture fluid, in different culture bottle, then add 2 μ LAd-SIRT1 adenoviral stocks (Ad-SIRT1 adenoviral stocks is so kind as to give by Zhu Weiguo professor) and Ad-GFP adenoviral stocks respectively, continue at 37 DEG C again, 5%CO
2incubator continues to cultivate 3-5 days, Microscopic observation infects the HEK293 cell of virus (owing to being infected by the virus, HEK293 cell rounding, bunchiness floats, there is cytopathic effect (cytopathic effect, CPE), can plaque be formed), then the HEK293 cell of virus will be infected from 37 DEG C, 5%CO
2incubator takes out and is put into-80 DEG C of refrigerator freezings, be put into 37 DEG C again to thaw, multigelation 3 times, makes lysis, release adenovirus, through hypervelocity refrigerated centrifuge under 10000r/pm condition after centrifugal 30min, with the membrane filtration of 4.5 μm, collect supernatant, be dispensed into 1mLEP pipe,-80 DEG C of cryogenic refrigerators are preserved, for subsequent use.
2. at Nanog
negnon-liver cancer stem cell in infect Ad-SIRT1 or Ad-GFP adenovirus
Get PLC/PRF/5 and Huh7 cell respectively, by fluidic cell sorting Nanog
negnon-liver cancer stem cell, and be seeded in 6 orifice plates, 37 DEG C, 5%CO
2incubator is cultivated, and treats that cell grows to 5 × 10
5time, liquid in sucking-off hole, changes the DMEM culture fluid of serum-free, and then every hole adds Ad-SIRT1 or Ad-GFP adenovirus with 10 MOI values, then at 37 DEG C, and 5%CO
2incubator cultivate, after 2 hours, liquid in sucking-off hole, change containing 10%FBS DMEM culture fluid continue cultivate, cultivate after 48 hours or 72 hours get final product testing goal gene expression or carry out subsequent experimental.
3. detect raise SIRT1 expression after Nanog
negon the impact of non-liver cancer stem cell
The albumen of the HEK293 cell of the Ad-SIRT1 adenovirus infection of extracting and contrast Ad-GFP adenovirus infection, then western blot method is utilized to detect the expression infecting 72h SIRT1 albumen, simultaneously with GAPDH albumen for internal reference, detection method is the same, and testing result is as shown in figure 12.As shown in Figure 12, relative to infection Ad-GFP adenovirus, raise infect Ad-SIRT1 in non-liver cancer stem cell after SIRT1 protein expression level significance.
In order to study the effect of SIRT1 in non-liver cancer stem cell further, detect the Nanog after infecting viral 72h
negclone formation, balling-up ability.Concrete grammar is: the Nanog being infected virus by Flow cytometry 200
negbe seeded in 24 orifice plates, arrange 3 repetitions, cultivate 14 days statistics cell clone quantity and cell balling-up ability, result as shown in figure 13.Result shows, at Nanog
negnon-liver cancer stem cell in, the formed clone of process LAN SIRT1 is larger than compared with control cells, and cloning efficiency significantly raises (PLC/PRF/5:P<0.048; Huh7:P<0.004), illustrate at Nanog
negnon-liver cancer stem cell in process LAN SIRT1 significantly can promote the ability of Clone formation.The statistical result of cell balling-up ability is shown, compared to compared with control cells, utilizes adenovirus process LAN SIRT1, can Nanog be raised
negballing-up ability (the PLC/PRF/5:P<0.033 of non-liver cancer stem cell; Huh7:P<0.027).Above result of study shows, process LAN SIRT1 can promote Nanog
negthe Clone formation of non-liver cancer stem cell and balling-up ability.
In order to study SIRT1 further to Nanog
negthe impact of tumor ability is grown into, at PLC/PRF/5Nanog in non-liver cancer stem cell body
neginfect respectively in Ad-SIRT1 or Ad-GFP and liver-cancer stem cell in non-liver cancer stem cell and infect shScramble, after 72h, the cell infecting adenovirus or slow virus is inoculated into NOD/SCID mice and detects its impact on tumor neoplasia ability, result as shown in figure 14.Result shows, and is respectively at shScramble, Ad-GFP and Ad-SIRT1 tumor formation rate of NOD/SCID mouse hypodermic inoculation equal number: 100%(10/10), 70%(7/10) and 100%(10/10) (Figure 14 A); Then statistics becomes tumor weight and volume after infecting, and becomes tumor weight to be respectively: 0.32414 ± 0.20g; 0.08445 ± 0.078g; 0.40645 ± 0.20g(Figure 14 B), gross tumor volume is respectively 0.061 ± 0.11cm
3; 0.016 ± 0.01cm
3; 0.04 ± 0.05cm
3(Figure 14 C).Above result shows at Nanog
negnon-liver cancer stem cell in process LAN SIRT1, significantly can raise the tumor formation rate of non-liver cancer stem cell and the volume of transplanted tumor and weight.
In sum, histon deacetylase (HDAC) SIRT1 has important function in liver-cancer stem cell self renewal.Therefore, the mechanism of action of SIRT1 in the regulation and control of liver-cancer stem cell self renewal, for deepening the cognition of liver-cancer stem cell and to develop new target medicine significant.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (7)
1. disturb SIRT1 to express the application of reagent in the medicine of preparation suppression liver-cancer stem cell self renewal.
2. application according to claim 1, is characterized in that: it is the slow virus that interference SIRT1 expresses that described interference SIRT1 expresses reagent.
3. application according to claim 2, is characterized in that: the slow virus that described interference SIRT1 expresses contains Lv-sh-SIRT1 plasmid.
4. application according to claim 3, is characterized in that: the slow virus that described interference SIRT1 expresses by after Lv-sh-SIRT1 plasmid transfection 293T cell, through packaging, purification and obtaining.
5. the application according to any one of claim 1-4, is characterized in that: interference SIRT1 expresses the application of reagent in the medicine of preparation suppression liver-cancer stem cell propagation.
6. the application according to any one of claim 1-4, is characterized in that: interference SIRT1 expresses the application of reagent in the medicine of preparation suppression liver-cancer stem cell Clone formation.
7. the application according to any one of claim 1-4, is characterized in that: interference SIRT1 expresses the application of reagent in the medicine of preparation suppression liver-cancer stem cell balling-up.
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