CN104774909A - Method for analysis of proanthocyanidins induced liver cancer cell autophagic death and application - Google Patents
Method for analysis of proanthocyanidins induced liver cancer cell autophagic death and application Download PDFInfo
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- CN104774909A CN104774909A CN201410555717.2A CN201410555717A CN104774909A CN 104774909 A CN104774909 A CN 104774909A CN 201410555717 A CN201410555717 A CN 201410555717A CN 104774909 A CN104774909 A CN 104774909A
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Abstract
The invention discloses a method for analysis of proanthocyanidins induced liver cancer cell autophagic death and application, and the human liver cancer cell HepG2 cell is used for experiment for determination of effects on HepG2 cell proliferation, dose effect and time effect of induction of HepG2 cell autophagic death of proanthocyanidins, content of intracellular ROS (reactive oxygen species and high active oxygen radicals), cell cycle distribution and mitochondrial membrane potential. Results confirm that the proanthocyanidins can induce human liver cancer cell HepG2 cell autophagic death by stimulating of the ROS pathway, and the results are applied to the prevention and treatment of liver cancer.
Description
Technical field
The present invention relates to medical art, be specifically related to a kind of pycnogenols induces the death of human liver cancer cell autophagy analytical procedure and novelty teabag based on active oxygen approach.
Background technology
Malignant tumour is one of three large diseases of serious harm human health, liver cancer is modal adult malignancies, and its mortality ratio occupies the 3rd of global cancer mortality, and still in rising trend, having more than 600,000 new cases every year, is still one of maximum disease threatening compatriots' life.Prevention or intervention are the effective means that clinical conventional suppression liver cancer worsens or reverses.But still there is many defects at present as the chemotherapeutics for the treatment of malignant tumor Main Means, as large in toxic side effect and tumour cell easily produces resistance etc.Seek good effect in recent years, toxic side effect is little, the drug candidate being played antihepatocarcinoma effect by multipath Mutiple Targets becomes global important research direction.The focus that the activeconstituents with antitumor action that is effective, low toxicity is world's antitumor drug research is always found from natural phant.
Pycnogenols (Procyanidins is called for short PC) is the large class phenol polymer be polymerized by catechin or l-Epicatechol.Such material has multiple biological activity, efficient with it, low toxicity and high bioavailability and famous, extensively be present in grape, hawthorn, peanut, the vegetables such as lotus pod, in fruit and nut, it is Recent study exploitation functional factor comparatively widely, pycnogenols has strong anti-oxidant activity as the free-radical scavengers of generally acknowledging, it is anti-oxidant is VE50 times with Scavenging ability, Vc20 doubly, and more than the 80 kind of disease caused because of free radical can be prevented, as cardiovascular system diseases, reducing blood-fat, radioprotective, anti-ageingly to wait for a long time, in addition anti-inflammatory is also had, anti-tumor activity etc.
China's Chinese chestnut cultivation history is long, and variety source enriches.Its fruit chestnut is the traditional agricultural byproducts of China, and annual production, more than 1,000,000 tons, accounts for 3/4 of world's Chinese chestnut ultimate production.But Chinese chestnut pulp is produced and in the course of processing, the chestnut shell of nearly 8.9-13.5% is still main goes out of use with burning and mode of naturally rotting, and only has minority to utilize chestnut shell to prepare the bibliographical information of gac, substratum, absorbing heavy metal ions in water and sterilant.And very few about chestnut shell chemical constitution study report, do not relate to the research of Chinese chestnut procyanidins (procyanidins from chestnut shell, CSPCs) and anti-tumor activity thereof.
Autophagy (Autophagic) be eukaryotic cell by Gene Handling by degraded himself tenuigenin and organoid carry out a series of programmed death processes of " self-digestion ".Autophagy is as the general and important multiple physiology of biological phenomena wide participation and pathologic process.Promote that cell survival again can the double mechanism of inducing cell initiative death because autophagy has, caused the great interest of domestic and international tumor research person.Since Levine in 2007 delivers and sets forth oncotherapy and autophagy dependency in autophagy and cancer one literary composition on " Nature ", autophagy has become the antitumor study hotspot of laying equal stress on apoptosis.
Summary of the invention
The object of this part is some aspects of general introduction embodiments of the invention and briefly introduces some better embodiment.May do in the specification digest and denomination of invention of this part and the application a little simplify or omit with avoid making this part, specification digest and denomination of invention object fuzzy, and this simplification or omit and can not be used for limiting the scope of the invention.
In view of the analytical procedure of above-mentioned and/or existing pycnogenols induction liver cancer cell autophagy death and pycnogenols Problems existing in treatment and the application in preventing liver cancer, propose the present invention.
Therefore, an object of the present invention is to provide the modeling and analysis methods of a kind of pycnogenols by the death of stimulating activity oxygen induction human liver cancer cell autophagy.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: the analytical procedure of a kind of pycnogenols induction liver cancer cell autophagy death, it comprises,
Pycnogenols is to the inhibiting analysis of liver cancer HepG2 growing multiplication:
The HepG2 cell of taking the logarithm vegetative period through tryptic digestion, after inoculation culture 24h, through pycnogenols process 6 ~ 120h, removing substratum also, after washing, adds MTT and hatches, and adds DMSO and dissolve after terminating, measure absorbancy in microplate reader 490nm place, calculate growth inhibition ratio;
CSPCs induces the dosage effect of HepG2 cell autophagy and the analysis of time effect:
Mark autophagocyte, after HepG2 cell inoculation culture is hatched 24h, with pycnogenols process cell different time or use rapamycin treatment 12h, after hatching 1h after experiment terminates, observes also fluorescent quantitation under inverted fluorescence microscope;
The analysis of transmission electron microscope observing cytolysosome:
Collect the HepG2 cell of each treatment group, centrifugal, abandon supernatant, fix respectively, then dewater, ultrathin section(ing), uranyl acetate and the plumbous negative staining of lemon, in transmission electron microscope observing also quantitatively;
The cycle analysis of cell:
Take the logarithm vegetative period HepG2 cell inoculation hatch 24h after, with pycnogenols process cell 12h, or hatch with pycnogenols after pretreatment, after experiment terminates, add the dyeing of iodate third ingot staining fluid lucifuge, the change of flow cytometer analysis of cells DNA content after filtering;
In cell, ROS (reactive oxygen species, highly reactive form of oxygen free radical) analyzes:
In cell, ROS content adopts oxidation-sensitive probe, DCFH-DA enters cell and has been oxidized to fluorescence 2'7'-dichlorofluorescein by ROS, the amount that ROS produces can be reflected by measuring DCF fluorescence intensity, cell is hatched with pycnogenols and positive control rapamycin respectively, hatch with pycnogenols subsequently, cell is after DCFH-DA process, also quantitative in fluorescence microscope immediately;
The analysis that mitochondrial membrane potential measures:
Mitochondrial membrane potential measures and adopts lipotropy fluorescent probe JC-1, in normal cell JC-1 with polymeric formal distribution in plastosome, red fluorescence can be inspired at 590nm place, show high membrane potential, when mitochondrial membrane potential declines, JC-1 is combined with film with monomeric form, green fluorescence is inspired at 530nm place, therefore the change of mitochondrial membrane potential can be judged by the red/green strength ratio of JC-1, by HepG2 cell through pycnogenols process 12h, harvested cell, washing, 30min is hatched in 37 DEG C with the perfect medium containing JC-1, directly analyze in fluorescence microscopy Microscopic observation,
Data Management Analysis:
Data represent with X ± SD, and adopt SPSS statistics software, carry out the process of t check data and variance analysis, wherein, X represents mean value, and SD represents standard deviation, whether there is significant difference between the population mean of certain variable of t representative inspection and certain designated value.
It is dead in treatment and the application in preventing liver cancer by stimulating activity oxygen induction human liver cancer cell autophagy that another object of the present invention is to provide a kind of pycnogenols.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: a kind of pycnogenols is dead in treatment and the application in preventing liver cancer by stimulating activity oxygen induction human liver cancer cell autophagy, and pycnogenols oligomer structural formula is:
Wherein, n=2 ~ 4.
As pycnogenols of the present invention by the dead a kind of preferred version in treatment and the application in preventing liver cancer of stimulating activity oxygen induction human liver cancer cell autophagy, wherein: described pycnogenols produces ROS induce liver cancer cell generation autophagy dead by stimulating in hepatoma Hep G 2 cells.
As pycnogenols of the present invention by the dead a kind of preferred version in treatment and the application in preventing liver cancer of stimulating activity oxygen induction human liver cancer cell autophagy, wherein: described pycnogenols is induced in the process of liver cancer cell generation autophagy death by stimulating generation ROS in hepatoma Hep G 2 cells, and the plastosome in liver cancer cell also take part in this process.
Beneficial effect of the present invention: the present invention adopts human hepatoma HepG2 cell to test, measure pycnogenols to the impact of HepG2 cell proliferation, the dosage effect of induction HepG2 cell autophagy death and time effect, ROS (reactive oxygen species in cell, highly reactive form of oxygen free radical) content, cell cycle distribution, mitochondrial membrane potential measures.Result confirms, pycnogenols induces human hepatoma HepG2 cell's autophagy dead by stimulating ROS approach, and by among this application of result to prevention and therapy liver cancer.
Accompanying drawing explanation
Fig. 1 is the restraining effect schematic diagram of CSPCs to proliferation of hepatocellular carcinoma HepG 2 cell line;
Fig. 2 is dosage effect and the time effect schematic diagram that CSPCs induces HepG2 cell autophagy, wherein, A is the fluorescence intensity change schematic diagram of CSPCs process cell after MDC dyeing, B and C is the fluorescence intensity schematic diagram that 300 μ g/mL CSPCs process different time MDC dye, D and E is LC3 level schematic diagram in 300 μ g/mLCSPCs process different time cells, F and G is MDC fluorescence intensity schematic diagram after CSPCs process cell 12h, H is various dose CSPCs process cell 12h LC3 level schematic diagram, I is the horizontal column schematic diagram of various dose CSPCs process cell 12h LC3,
Fig. 3 is transmission electron microscope observation HepG2 cellular form (6600 ×) schematic diagram, and wherein, A is control group schematic diagram, B is 3-MA treatment group schematic diagram, C be in advance after 3-MA process 1h with 300 μ g/mL CSPCs treatment group schematic diagram, D is positive controls, i.e. 1nmol/L rapamycin treatment 12h schematic diagram, E is 300 μ g/mL CSPCs process cell 12h schematic diagram, in figure, arrow instruction cytolysosome, F is cytolysosome quantitative analysis schematic diagram, in figure, compared with E group
*p < 0.005;
Fig. 4 is autophagocytosis (400 ×) schematic diagram that CSPCs induces HepG2 cell, wherein, A is control group schematic diagram, B is positive controls, i.e. 1nmol/L rapamycin treatment 12h schematic diagram, C is 300 μ g/mL CSPCs process 12h schematic diagram, and D is that the fluorescence intensity of MDC evaluates autophagy ratio schematic diagram, E and F is LC3-II protein expression and LC3-II/β-actin ratio schematic diagram;
Fig. 5 is autophagy (400 ×) schematic diagram that 3-MA pre-treatment reduces that CSPCs induces HepG2 cell, wherein, A is control group schematic diagram, and B is 300 μ g/mL CSPCs group schematic diagram, C be in advance after 3-MA process 1h with 300 μ g/mL CSPCs treatment group schematic diagram, D is 3-MA group schematic diagram, E is fluorescent quantitation schematic diagram after MDC dyeing, and F is LC3-II protein expression schematic diagram, and G is Average LC3-II/actin ratio schematic diagram, wherein
*p < 0.005 is compared with A group;
*p < 0.005 is compared with C group;
Fig. 6 is that CSPCs induces the HepG2 cell S phase to block schematic diagram, and wherein, A is control group schematic diagram, B to be 300 μ g/mL CSPCs group schematic diagram C be in advance after 3-MA process 1h with 300 μ g/mL CSPCs treatment group schematic diagram, D is 3-MA group schematic diagram;
Fig. 7 is that CSPCs stimulates HepG2 cell to produce ROS schematic diagram, wherein, A is the schematic diagram of CSPCs process cell different time ROS fluorescence intensity, B produces ROS schematic diagram through CSPCs process 12h dose-dependant, C is ROS fluorescence intensity schematic diagram after various dose CSPCs process cell 12h, and wherein * P<0.05 is compared with control group.
Fig. 8 is after 5mmol/L N-acetylcystein (Nac) pre-treatment 2h, again through 300 μ g/mL CSPCs process 12h on the schematic diagram of the impact of the cell autophagy death that CSPCs induces, wherein, A is MTT analysis of cells existence inhibiting rate schematic diagram, B is LC3-II protein expression schematic diagram, C is that LC3-II/ β-actin expresses ratio schematic diagram, and wherein * P<0.05 compared with the control;
#p<0.05 and Nac+CSPCs group is compared;
Fig. 9 is JC-1 dyeing schematic diagram after 300 μ g/mL CSPCs process HepG2 cell 12h, and wherein, A is JC-1 fluorescent staining figure, B is red/green fluorescence strength ratio figure after JC-1 dyeing, and wherein * P<0.05 has significant difference compared with control group; * P<0.01 has pole significant difference compared with control group.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail below by the specific embodiment of the present invention.
Set forth a lot of detail in the following description so that fully understand the present invention, but the present invention can also adopt other to be different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar popularization when intension of the present invention, therefore the present invention is by the restriction of following public specific embodiment.
Carry out inducing the activity of human hepatoma HepG2 cell autophagy death and ROS approach to inquire into chestnut shell pycnogenols below, its novelty teabag in pharmacy and field of health care products is described.
Through measuring, the pycnogenols oligomer structural formula being applied to prevention and therapy liver cancer field is:
Wherein, n=2 ~ 4.
Embodiment 1 pycnogenols is dead based on active oxygen approach induction human hepatoma HepG2 cell autophagy
1 experiment material
1.1 test sample
Chestnut shell is pulverized, add the sodium bisulfite of 0.05 ‰ ~ 0.1 ‰, lixiviate 0.3-1h is sealed in boiling water, vat liquor 200 order screen filtration, the macroporous adsorbent resin AB-8 post that filtrate flowing through is anticipated, after drawing non-adsorbable impurity with deionized water, with the ethanol-eluting resin column of 70%, obtain ethanol eluate, less than 45 DEG C decompression recycling ethanols, lyophilize or spraying dry is adopted to obtain light brown chestnut shell pycnogenols (procyanidins from chestnut shell, CSPCs).Be made into perfect medium in experimentation and need concentration, matching while using.
1.2 human hepatoma HepG2 cell
Be purchased from Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences.Cell is with DMEM substratum (10% newborn calf serum and 100 μ g/mL Streptomycin sulphates and 100U/mL penicillin).Until cell grows to 80% fusion, trypsin digestion and cell (0.5% trypsinase/2.6mM EDTA), uses PBS wash-out.
2 experimental techniques
2.1 pycnogenolss are to the restraining effect of liver cancer HepG2 growing multiplication
The HepG2 cell of taking the logarithm vegetative period, after tryptic digestion, is inoculated in (2.0 × 103 cells/well) in 96 orifice plates, in 37 DEG C, 5%CO
2, cultivate 24h under saturated humidity after, process 6-120h through the CSPCs (0-400 μ g/mL) of different concns.Cultivation terminates rear absorption substratum, after washing 2 times, adds the MTT (1mg/mL) of 100 μ L with PBS, 4h is hatched in 37 DEG C, add 150 μ L DMSO after end and dissolve 10min, measure absorbancy (Multiskan MK3, USA) in microplate reader 490nm place.Calculate growth inhibition ratio as follows: inhibiting rate (%)=(A
control group-A
cSPCs)/A
control group× 100.
2.2CSPCs induces dosage effect and the time effect of HepG2 cell autophagy
Monodansylcadaverine (MDC) is adopted to mark autophagocyte.HepG2 cell is inoculated in (1 × 105 cell/every hole) in 6 well culture plates, in 37 DEG C, 5%CO
2, hatch 24h under saturated humidity after, process 12h with the CSPCs process cell different time of different concns or 1nmol/L rapamycin (positive drug Rapamycin), or hatch 12h with CSPCs after 1mM 3-MA pre-treatment 1h.After experiment terminates, after hatching 1h with 0.05mM MDC 37 DEG C, observe and fluorescent quantitation under inverted fluorescence microscope (Leica DMI 4000B, Germany).Its fluorescence intensity (%)=(treatment group fluorescence intensity-control group fluorescence intensity)/control group fluorescence intensity × 100.
2.3 transmission electron microscope observing cytolysosomes
Collect the HepG2 cell of each treatment group, centrifugal, abandon supernatant, fix with the PBS containing 3% glutaraldehyde and the PBS containing 1% perosmic anhydride respectively.Then with graded ethanol dehydration, ultrathin section(ing), uranyl acetate and the plumbous negative staining of lemon, observe also quantitatively in transmission electron microscope (TEM) (Philips, Holland).Cytolysosome=treatment group cytolysosome mean value/control group mean value.
2.4 cell cycle analysis
The vegetative period HepG2 cell of taking the logarithm to be inoculated in 6 well culture plates in (1 × 105 cell/every hole), in 37 DEG C, 5%CO
2, hatch 24h under saturated humidity after, with 300 μ g/mL CSPCs process cell 12h, or hatch 12h with CSPCs after 1mM 3-MA pre-treatment 1h.After experiment terminates, add iodate third ingot staining fluid (containing RNA enzyme) lucifuge dyeing 30min, the change of flow cytometer analysis of cells DNA content after 300 order nylon net filters.
In 2.5 cells, ROS analyzes
In cell, ROS content adopts oxidation-sensitive probe (2', 7'-dichlorofluorescein diacetate, DCFH-DA), DCFH-DA enters cell and has been oxidized to fluorescence 2'7'-dichlorofluorescein (DCF) by ROS, can reflect by measuring DCF fluorescence intensity the amount that ROS produces.Cell is hatched 12h with 300 μ g/mL CSPCs and 1nmol/L positive control rapamycin respectively or in advance with 1mM 3-MA process 1h, hatches 12h with CSPCs subsequently.Cell is after 5 μMs of DCFH-DA process 30min subsequently, immediately in fluorescence microscope also quantitatively.
2.6 mitochondrial membrane potentials measure
Mitochondrial membrane potential measures and adopts lipotropy fluorescent probe JC-1.In normal cell, JC-1 is with polymeric formal distribution in plastosome, can inspire red fluorescence at 590nm place, shows high membrane potential.When mitochondrial membrane potential declines, JC-1 is combined with film with monomeric form, inspires green fluorescence at 530nm place, therefore can be judged the change of mitochondrial membrane potential by the red/green strength ratio of JC-1.By HepG2 cell through CSPCs process 12h, harvested cell, washes twice, hatch 30min with containing the perfect medium of 10 μ g/mL JC-1 in 37 DEG C, directly analyzes in fluorescence microscopy Microscopic observation.
2.7 data processing
Data represent with X ± SD, adopt SPSS statistics software, and carry out the process of t check data and variance analysis wherein, X represents mean value, and SD represents standard deviation, whether there is significant difference between the population mean of certain variable of t representative inspection and certain designated value.
3 experimental results
3.1CSPCs is on the impact of proliferation of hepatocellular carcinoma HepG 2 cell line
MTT method evaluation CSPCs to the result of HepG2 cell proliferation as shown in Figure 1.There is dosage and time-dependent relation in the restraining effect of CSPCs to HepG2 cell proliferation.CSPCs process cell 90h to 120h in 25-400 μ g/mL concentration range, to the inhibiting rate of HepG2 cell respectively from (6.3 ± 1.16) % to (91.3 ± 1.41) % and the IC50 of (7.5 ± 1.78) % to (91.9 ± 1.64) %, CSPCs effect HepG2 cell 6-120h between (157.5 ± 0.57) μ g/mL and (84.55 ± 0.4) μ g/mL.
3.2CSPCs induces the effect of HepG2 cell autophagy
MDC is the effective fluorescence dye observing autophagy vesicle, can pass through MDC fluorescence intensity observation of cell autophagy level.As shown in Figure 2 A, within the scope of experimental concentration, CSPCs increases MDC fluorescence intensity with dose dependent manner for the time effect of CSPCs induction autophagy and dosage effect.300 μ g/mL CSPCs groups, when processing cell 12h, drop to (39.68 ± 8.74) % when its MDC fluorescence intensity can reach maximum value (71.69 ± 9.61) %, 36h.
In order to better study the effect of dosage and time, MDC fluorescence intensity and LC3-II expression amount is observed respectively with 50,100,150 and 300 μ g/mL CSPCs effects 6,12,24 and 36h, its result is as shown in Fig. 2 B, C, F and G, 300 μ g/mL CSPCs pre-treatment 12h group HepG2 cells show strong fluorescence, and can be observed MDC spot distribution.
Western blotting result is as shown in Fig. 2 D, E, H and I, and LC3-II/ β-actin ratio strengthens along with the prolongation in CSPCs treatment time and the increase of concentration, and can increase LC3-I after CSPCs process and change to LC3-II.LC3-II is the labelled protein of autophagy, LC3 has two types, a kind of is the cytoplasmic protein (LC3-I) of unprocessed 18kDa and the 16kDa albumen (LC3-II) of processing albumen, and LC3-II is combined in cytolemma, and can be converted by LC3-I during autophagy.Result shows, CSPCs can induce hepatoma Hep G 2 cells autophagy.
3.33-MA part suppresses the autophagy of CSPCs induction
This experiment selects 300 μ g/mL CSPCs effect HepG2 cell 12h best activity and time to carry out follow-up study.The typical duplicature of transmission electron microscope observing autophagic vacuole and content (Fig. 3 A).300 μ g/mL CSPCs process cell 12h, have cavity in observation of cell, also containing organoid and material to be digested in cavity, show that CSPCs can induce HepG2 cell autophagy body to produce (as Fig. 3 E).Positive control rapamycin group HepG2 cell also produces autophagic vacuole (Fig. 3 D).But through CSPCs process before 3-MA adds cell, find that cell autophagy bubble tails off (Fig.3C and F), show that 3-MA can suppress autophagy to occur really, weaken the autophagy generation of CSPCs induction.
MDC fluorescence intensity increases cell autophagy and increases, as Fig. 4.(Fig. 4 A) CSPCs treatment group (Fig. 4 C) cell MDC fluorescence intensity increases compared with control group.Compared with the control, 300 μ g/mL CSPCs process cell 12h show high MDC fluorescence intensity (61.82 ± 3.06%) (Fig. 4 D), and LC3-II protein expression increases (Fig. 4 E and F), shows similar result to positive control 1nmol/L rapamycin.As compared to CSPCs group fluorescence intensity (61.82 ± 3.06%) (Fig. 5 B with E), 3-MA pretreated group fluorescence intensity is that (40.64 ± 1.36%) (Fig. 5 C and E) and LC3-II albumen (Fig. 5 F and G) expression all reduce.With control group fluorescence intensity (27.64 ± 3.24%) (Fig. 5 A and E) and 3-MA fluorescence intensity (25.92 ± 2.99%) (Fig.5D and E), 300 μ g/mL CSPCs group fluorescence intensities are higher.Microtubule bindin LC3 induction autophagy after is combined with cytolysosome film, so cytolysosome formed and LC3-II be expressed as positive correlation.Above-mentioned data declaration 3-MA suppresses CSPCs-to induce HepG2 cell autophagy.
3.4CSPCs induces the S phase to block
In order to study the impact that CSPCs synthesizes DNA, the cell cycle distribution of CSPCs to HepG2 cell is studied.As shown in Figure 6, CSPCs function cells is after 12 hours, and the ratio of S phase cell increases, and the G0/G1 phase reduces (Fig. 6 A and B) accordingly simultaneously.Compared with CSPCs effect group, add CSPCs group after 3-MA pre-treatment and obviously decline in S phase Arrested Cell ratio.Table 1 shows, after CSPCs acts on 12 hours, G0/G1 phase cell (28.84%) significantly reduces, and the cell of 63.16% is still at S phase (Fig. 6 B).Meanwhile, after 300 μ g/mL CSPCs process cells, S phase cell rises to 63.16% (Fig. 6 A and B) from 25.41%.Be 53.71% (Fig. 6 C) with adding CSPCs group S phase cell after 3-MA pre-treatment.Show the synthesis that have impact on DNA in CSPCs Induces Autophagy process.
Table 1 CSPCs is to the percentile change of HepG2 cell each cell cycle
*p<0.05 is compared with control group
The autophagy of 3.5CSPCs induction produces relevant with ROS
ROS can be used as signaling molecule can activating cells autophagy.After this research finds CSPCs process HepG2 cell 6h, DCF fluorescence intensity increases, and showing that ROS produces increases, and the generation of ROS and CSPCs be lifetime effect relation (Fig. 7 A) not, but there is dose-dependent relationship (Fig. 7 B).Fig. 7 B and C shows 300 μ g/mL CSPCs process 12h, and ROS reaches maximum value in cell, has significant difference (P<0.05) compared with control group.Meanwhile, LC3-II protein expression level also increases (Fig. 2) with CSPCs action time, shows that ROS take part in autophagy process.
Now there are some researches show through active oxygen blocker N-acetylcystein (NAC) pre-treatment, the increase of ROS can be suppressed and lower cell mortality.Therefore, this experiment adopts the impact that ROS blocking-up NAC investigation CPSCs produces ROS.Result shows, compared with CSPCs group, the pretreated cells survival inhibiting rate of NAC reduces, the expression of LC3-II also reduces (Fig. 8 A, B and C), the toxicity of NAC+CSPCs treatment group to HepG2 cell also reduces, and these results show that CSPCs mediates autophagy and may depend on its generation ROS ability.
Mitochondrial membrane potential measurement result shows, compared with control group, CSPCs pretreated group JC-1 red/green fluorescence intensity significantly declines (P<0.01).Compared with CSPCs individual curing group autophagy inhibitor 3-MA pretreated group red/green fluorescence intensity enhancing, 3-MA+CSPCs group is red/green fluorescence intensity also increases (Fig. 9 A) to some extent.As Fig. 9 B shows, the cell of CSPCs process red/green fluorescence intensity be 3.55 ± 0.95,3-MA+CSPCs group red/green fluorescence intensity is 4.15 ± 0.71, shows the autophagy approach that the loss of mitochondrial membrane potential may participate in ROS and triggers.
This research confirms that CSPCs is dead by autophagy induction human HepG2 cell.CSPCs induces HepG2 cell autophagy to be by ROS approach, and autophagocytosis can part be blocked by ROS blocker Nac.Autophagy defines the autophagy vesicle of typical duplicature, observes the expression of autophagy fluorescence intensity and LC3 and LC3-I to the transformation of LC3-II, study CSPCs and induce HepG2 cell autophagy death effect by MDC dyeing.The distribution of cell cycle is have impact in CSPCs Induces Autophagy process.In addition, CSPCs group cell membrane potential significantly reduces compared with 3-MA group.These results hint CSPCs produces induction HepG2 cell autophagy by stimulating ROS, may be relevant with the signal pathway that plastosome relies on.
As can be seen here, the invention provides a kind of pycnogenols and produce by stimulating activity oxygen the purposes that preparation suppresses in the medicine of liver cancer cell autophagy death and protective foods.Be specifically related to CSPCs to the impact of hepatoma Hep G 2 cells viability, the dosage effect of CSPCs induction HepG2 cell autophagy and Time-effect relationship, autophagy inhibitor 3-MA (3-Methyladenine, be called for short 3-MA) on the impact of CSPCs Induces Autophagy, the impact of CSPCs Induces Autophagy cell cycle, ROS inhibitor N-acetylcystein (N-Acetyl-cysteine, be called for short Nac) on the impact of CSPCs Induces Autophagy and and the relation that produces of ROS, inquire into the approach that pycnogenols induces the death of liver cancer cell autophagy.
It should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (4)
1. an analytical procedure for pycnogenols induction liver cancer cell autophagy death, is characterized in that: comprise,
Pycnogenols is to the inhibiting analysis of liver cancer HepG2 growing multiplication:
The HepG2 cell of taking the logarithm vegetative period through tryptic digestion, after inoculation culture 24h, through pycnogenols process 6 ~ 120h, removing substratum also, after washing, adds MTT and hatches, and adds DMSO and dissolve after terminating, measure absorbancy in microplate reader 490nm place, calculate growth inhibition ratio;
CSPCs induces the dosage effect of HepG2 cell autophagy and the analysis of time effect:
Mark autophagocyte, after HepG2 cell inoculation culture is hatched 24h, with pycnogenols process cell different time or use rapamycin treatment 12h, after hatching 1h after experiment terminates, observes also fluorescent quantitation under inverted fluorescence microscope;
The analysis of transmission electron microscope observing cytolysosome:
Collect the HepG2 cell of each treatment group, centrifugal, abandon supernatant, fix respectively, then dewater, ultrathin section(ing), uranyl acetate and the plumbous negative staining of lemon, in transmission electron microscope observing also quantitatively;
The cycle analysis of cell:
Take the logarithm vegetative period HepG2 cell inoculation hatch 24h after, with pycnogenols process cell 12h, or hatch with pycnogenols after pretreatment, after experiment terminates, add the dyeing of iodate third ingot staining fluid lucifuge, the change of flow cytometer analysis of cells DNA content after filtering;
In cell, highly reactive form of oxygen free radical is analyzed:
In cell, highly reactive form of oxygen free-radical contents adopts oxidation-sensitive probe, DCFH-DA enters cell and is become to have fluorescence 2'7'-dichlorofluorescein by highly reactive form of oxygen free-radical oxidn, the amount that highly reactive form of oxygen free radical produces can be reflected by measuring DCF fluorescence intensity, cell is hatched with pycnogenols and positive control rapamycin respectively, hatch with pycnogenols subsequently, cell is after DCFH-DA process, also quantitative in fluorescence microscope immediately;
The analysis that mitochondrial membrane potential measures:
Mitochondrial membrane potential measures and adopts lipotropy fluorescent probe JC-1, in normal cell JC-1 with polymeric formal distribution in plastosome, red fluorescence can be inspired at 590nm place, show high membrane potential, when mitochondrial membrane potential declines, JC-1 is combined with film with monomeric form, green fluorescence is inspired at 530nm place, therefore the change of mitochondrial membrane potential can be judged by the red/green strength ratio of JC-1, by HepG2 cell through pycnogenols process 12h, harvested cell, washing, 30min is hatched in 37 DEG C with the perfect medium containing JC-1, directly analyze in fluorescence microscopy Microscopic observation,
Data Management Analysis:
Data represent with X ± SD, and adopt SPSS statistics software, carry out the process of t check data and variance analysis, wherein ,-X represents mean value, and SD represents standard deviation, whether there is significant difference between the population mean of certain variable of t representative inspection and certain designated value.
2. pycnogenols is dead in treatment and the application in preventing liver cancer by stimulating activity oxygen induction human liver cancer cell autophagy, it is characterized in that: pycnogenols oligomer structural formula is:
Wherein, n=2 ~ 4.
3. pycnogenols as claimed in claim 2 is dead in treatment and the application in preventing liver cancer by stimulating activity oxygen induction human liver cancer cell autophagy, it is characterized in that: to produce highly reactive form of oxygen free yl induction liver cancer cell generation autophagy dead by stimulating in hepatoma Hep G 2 cells for described pycnogenols.
4. pycnogenols as claimed in claim 3 is dead in treatment and the application in preventing liver cancer by stimulating activity oxygen induction human liver cancer cell autophagy, it is characterized in that: described pycnogenols is by stimulating in the process of generation highly reactive form of oxygen free yl induction liver cancer cell generation autophagy death in hepatoma Hep G 2 cells, and the plastosome in liver cancer cell also take part in this process.
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| CN105838354A (en) * | 2016-04-26 | 2016-08-10 | 中国科学院化学研究所 | Application of HQO as fluorescent probe used for monitoring mitochondrial autophagy process in live cells |
| CN110368381A (en) * | 2019-08-06 | 2019-10-25 | 扬州大学 | Application of the black fruit fructus lycii anthocyanidin in the drug that preparation is used to induce two kinds of cancer cell-apoptosis |
| CN113440505A (en) * | 2021-06-16 | 2021-09-28 | 东北农业大学 | Application of N-acetylcysteine in preparation of medicine for treating pig intestinal injury caused by glyphosate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105838354A (en) * | 2016-04-26 | 2016-08-10 | 中国科学院化学研究所 | Application of HQO as fluorescent probe used for monitoring mitochondrial autophagy process in live cells |
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| CN113440505A (en) * | 2021-06-16 | 2021-09-28 | 东北农业大学 | Application of N-acetylcysteine in preparation of medicine for treating pig intestinal injury caused by glyphosate |
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