Diffusa on cervical cancer Hela cell cycle apoptosis and telomerase activity.doc

1、 Diffusa on cervical cancer Hela cell cycle apoptosis and telomerase activity Of: superb, Liu Ying, Cai Xiaomin, Hao Xingzhi Abstract Objective To study the traditional Chinese medicine diffusa (Hedyotic diffusa, HD on telomerase activity in human cervical carcinoma Hela cells, cell cycle and apopto

2、sis, to investigate the inhibition of Hela cells in HD tumor activity and telomerase. Methods concentration of each of the different role of Hela cells in vitro, using PCR-TRAP-ELISA Quantitative Detection of HD treatment Hela cells telomerase activity level, the synchronization of cell cycle detect

3、ed by flow cytometry and the apoptosis rate of change . Results Hela cells were positive for telomerase, the role of a certain concentration of HD after a certain time in Hela cells can Hela cells were arrested in S phase and induce apoptosis, while telomerase activity was significantly reduced. Con

4、clusions may be adopted HD Hela cell cycle changes (S phase arrest and also induced apoptosis, decreased telomerase activity to achieve anti-tumor effect. Keywords: diffusa, cervical cancer Hela cells, cell cycle, apoptosis, telomerase In recent years some scholars diffusa (Hedyotic diffusa, HD anti

5、-tumor effects have been studied, its essential to be sure that anti-tumor effect 1-4, but its mechanism of action, especially not in-depth understanding of the molecular biological mechanisms. Telomerase expression and apoptosis in tumors and play an important role in the development, how to induce

6、 tumor cell apoptosis and regulation of telomerase activity in cancer treatment has become a new way. HD vitro and in vivo has obvious tumor effect, whether this effect induced tumor cell apoptosis, cell cycle arrest and inhibition of telomerase activity, not more than reported in literature. In thi

7、s study, human cervical carcinoma Hela cells were studied, using PCR products of polypropylene amide gel electrophoresis and silver staining qualitative detection of telomerase activity, PCR-TRAP-ELISA method for quantitative analysis of Hela cells after HD treatment levels of telomerase activity, s

8、imultaneous use of flow cytometry cell cycle and apoptotic rate, Anti-tumor mechanism of HD. 1 Materials and methods 1.1 Materials 1.1.1 Hela cell culture Hela cells from the CAS Shanghai Institute of Cell Biology, Cell Bank provides, as a culture medium containing 10% fetal bovine serum, 100 103 U

9、/ L penicillin, 0.1 g / L streptomycin in RPMI 1640, with a 100 ml flask, set 5 % CO2, 37 , humidity incubator saturated culture, cells reached 80% to 90% confluence state with 0.25% trypsin digestion passage. keep freezing during the experiment, all experiments were performed using exponential phas

10、e cells. 1.1.2 Reagents Fetal bovine serum (Tianjin Blood Institute), trypsin, ribonuclease (RNase), propidium iodide (PI, PCR-TRAP-ELISA telomerase detection kit were purchased from Sino-American Biotechnology Company. 1.1.3 Drugs HD (Jiangsu Jiangxi Fearless Chinese pharmaceutical group I) 200 g 2

11、 000 ml distilled water boiling temperature of the fire 60 min, boiled 30 min, sieve filters to Java, the filtrate was centrifuged 20 min, the supernatant was concentrated to 200 ml, 0.22 m filter sterilization, was 1 kg / LHD extract use. 1.2 Experimental Methods 1.2.1 Drug Treatment 0.25% trypsin

12、digestion, with RPMI 1640 medium to adjust cell density of 1 108 / L, the increase each flask 1 ml, then the culture medium by adding 6 ml, set 5% CO2, 37 constant temperature incubator cultured for up to 70% of students to be fused cells replace the culture medium while adding HD extract, final con

13、centration of drug 20,40,80 g / L, different concentrations of the same culture time. set equal volume of culture medium for the experimental group were . 1.2.2 Flow cytometry (FCM) analysis of cell cycle and apoptosis in Hela Collection, including the treatment of Hela adherent and suspension cells

14、, cell density was adjusted with PBS 1 109 / L, take 1 ml single cell suspension, centrifugation, washing, 1 ml 70% ethanol fixed cells, and then PBS rinsing, the samples in a single cell by adding RNase, then add a final concentration of 50 g / L of PI staining, measured after filtration with a FCM

15、. to normal peripheral blood lymphocytes for the normal diploid standard, the determination of DNA based on the Prescription map analysis software with CELLQUEST Hela cell cycle and apoptotic index. 1.2.3 Detection of telomerase activity 1.2.3.1 Bradford protein determination of the concentration of

16、 cell extracts Bradford method of protein and production of optical density standard curve, according to wavelength of the samples under 595 nm optical density (D, read from the standard curve of the sample protein extract protein concentration. 1.2.3.2 PCR-TRAP qualitative detection of silver stain

17、ing Take 20l PCR product mixture by adding 4 l gel sample buffer, and set the positive and negative control of telomerase activity, the sample was 12.5% polyacrylamide gel electrophoresis separation. Will be placed in silver staining gel stationary oscillation gently 30 min, washed 2 to 3 times afte

18、r gel into staining solution in 0.2% silver nitrate and then transferred to color liquid gel (3% sodium hydroxide, 0.3% formaldehyde until the brown yellow bands appear, at 5% acetic acid stop solution, the final preservation solution on the gel (10% glycerol, 2% ethanol, soaked in 60 min, UVP Adhes

19、ive print or save the system analysis. 1.2.3.3 PCR-TRAP-ILISA quantitative detection Take TRAP Tubes, all joined the TRAP Mix 45 l, cell extracts 2.0 l (protein content of 0.5 10 g), mix, covered Paraffinliquid 30 l, centrifuge a few seconds, set 25 water bath for 30 min, PCR amplification of the li

20、ne . obtained PCR product 25 l, adding Hyb Reagent A 25 l, after mixing, 25 l were taken to the hole, mix, set the positive and negative control of telomerase activity, constant temperature water bath at 37 for 60 min reaction . each hole by adding Chromogen A, Chromogen B of the 50 l, 37 dark react

21、ion of 10 min, by adding Stop Solution 100 l, terminate the reaction. microplate reader to read on the wavelength of 450 nm D value. sample value is greater than or equal to D negative control, 2.1 times the D value of telomerase activity was positive. 1.3 Statistical analysis Application of SPSS 6.

22、0 statistical software for statistical analysis, measurement data were +-s that differences between groups using t test, test = 0.05 level. 2 Results 2.1 HD on Hela cell cycle and apoptosis Extracts with different concentrations of HD action time increase in Hela cells, G0/G1 cells decreased, S phas

23、e cells increased, while the G2 / M cells did not change. The results shown in Table 1. Table 1 Flow cytometry HD effect 48 h Hela cell cycle and apoptosis results (omitted) 2.2 HD on the Hela cell line telomerase activity 2.2.1 PCR-TRAP silver staining results of the expression of telomerase-positi

24、ve Hela cell line for silver staining after 6 bp intervals brown bands, 80 g / L after 48 h, the concentration of HD telomerase still on Hela cells positive, but electrophoresis to determine its activity on the decline. Figure 1. Links to free paper download 2.2.2 PCR-TRAP-ELISA quantitative test re

25、sults 20,40,80 g / L HD role of 48 h, telomerase activity has declined .20 g / L in the HD effect, each time point compared with the experimental control group were not significantly different (P> 0.05, 40 g / L and 80 g / L in the HD effect on Hela cells in 48 h, telomerase activity was signific

26、antly decreased compared with the control group was significantly different (P <0.01. Figure 2. comparative silver staining PCR-TRAP result, the higher the value of quantitative detection of D results of silver staining greater the number of bands, the higher the resolution. 3 Discussion Plants i

27、n recent years to find effective and side effects of anticancer drugs has become an important issue at home and abroad, on the antitumor mechanism of traditional Chinese medicine in the ascendant. HD bears the chemical composition of the major acid, immune polysaccharides, ursolic acid, HD Su and ot

28、her, studies have shown HD has some antitumor activity against a variety of in vitro growth inhibition of tumor cell lines 2-4. flow cytometry measured the role of a certain concentration of HD Hela cells after a certain time, there may be sub- diploid apoptotic peak, 80 g / L HD induced Hela cell a

29、poptosis rate was 21.34%, and the experimental control group showed a difference compared the natural apoptosis rate was significantly (P <0.01. so that in a certain range of dose and time , HD Hela cells can inhibit growth and induce apoptosis in Hela cells may be one of the mechanisms. Studies

30、have shown that cell proliferation and apoptosis in the incidence of malignant tumors on the closely related process of blocking the cell cycle can cause apoptosis, while apoptosis was usually associated with growth inhibition, cell cycle and apoptosis are closely related. Some scholars believe that

31、 G0/G1 phase cells was only apoptosis, but more scholars believe that the cell cycle phases of apoptosis can occur, different treatment and different factors have different cell cycle specificity of apoptosis 5. cell proliferation and subject to G1 S G2 M 4 periods, the biosynthesis of any phase may

32、 lead to apoptosis is blocked. In this study, flow cytometry cell cycle analysis, results suggest that HD can Hela cells in G0 / reduce the proportion of cells in G1, S phase cells was increased, indicating that the HD to Hela cells were arrested in S phase, S phase was extended to a certain period

33、of time after the loss of proliferation leading to apoptosis. The results suggest that clinical treatment of malignant tumors using the HD at the same time, the choice of S phase-sensitive chemotherapy drugs to enhance therapeutic effect. Telomerase is a cellular RNA dependent DNA polymerase, to its

34、 own RNA as a template telomerase reverse transcriptase synthesis of DNA, on an important role in maintaining telomere length, telomerase to prevent telomere shortening with high expression and the promotion of cancer tissue cloning, reproduction, development. has been found that 80% to 90% of human

35、 malignant tumors can be detected in telomerase-positive, and most normal somatic cells do not express telomerase activity 6-8. Malignant Tumors The high expression of telomerase, it has a higher ability of anti-apoptosis, a number of chemically induced apoptosis of tumor cells may be related to inh

36、ibition of telomerase activity within cells 9-11. According to statistics, more than 90% even up to 100% of cervical cancer can be detected in the expression of telomerase. The experimental results show that a certain concentration of HD in the induction of apoptosis in Hela cells also accompanied b

37、y decreased telomerase activity, with the HD concentration and time extension of telomerase activity in Hela cells decreased, while the cells the rise of apoptosis. The results suggest that, HD cells, telomerase activity can be reduced, Hela cell apoptosis may be related to HD down telomerase activi

38、ty. in-depth study will help further understanding of the relationship between the two anti-tumor mechanism of HD and inhibition of telomerase activity and induction of apoptosis for the treatment of malignant tumors in this new way to provide a theoretical basis, and contribute to development in or

39、der to inhibit telomerase activity and induction of apoptosis as a target for anti-tumor medicine. References 1 Bao-En, Zhang Jinyan, Duxiao Na, et al. Diffusa regulate immunological activity and antitumor activity J. Journal of Traditional Chinese Medicine, 2001,21 (5:370-374. 2 Chen Liping, Yang X

40、iangsheng, Wei Xing, et al. Diffusa extract induced lung SPC-A-1 cell apoptosis in experimental study J. Jiangxi Medical College, 2005,17 (6:53-55 . 3 in the Chun-yan, Li Wei, Liu, and, and so on. Diffusa multidrug resistance in human liver cancer cells Bel-7402 anti-tumor activity J. North China University of Technology: Natural Science, 2004, 5 (3:221-223. 4 Li R, Zhao HR, Lin YH. Anti-tumor effect and protective effect on chemotherapeutic damage of water soluble extracts from hedyotis diffusa J. Journal of Chinese Pharmaceutical Sciences, 2002,11 (2) :54-58. 5 SrivastavaJ