不同部位角膜上皮细胞体外增殖性的对比研究

为观察角膜缘、角膜周边及角膜中央部上皮细胞体外增殖的规律，确定角膜干细胞以选取体外培养角膜上皮细胞的部位。应用溴代脱氧尿嘧啶（Ｂｒｄｕ，胸腺嘧啶的类似物）掺入ＤＮＡ合成，抗－Ｂｒｄｕ单克隆免疫荧光抗体记标记细胞法，在流式细胞仪（ＦＡＣＳｔａｒｐｌｕｓ）上检测体外培养的每一代角膜中央部、周边部及角膜缘上皮细胞增殖的情况。结果显示从第一代开始，角膜缘角膜周边角膜中央部上皮细胞增殖率逐渐递减；角膜缘、角膜周边、角膜中央部细胞代数依次减少；原代角膜缘及角膜周边部上皮细胞增殖率经统计学处理，无显著性差异（Ｐ＞００５）；结论：（１）角膜缘部细胞增殖潜力大；（２）角膜缘上皮细胞群中存在一定数量的长周期细胞。因此，角膜缘有角膜干细胞存在。

Proliferation inhibition of human cervical cancer HeLa cells by Casticin in vitro

Objective： The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods： Human cervical HeLa cells were cultured in vitro. The inhibitory effect of Casticin on the viability of human cervical cancer HeLa ceils was evaluated by the MTT assay. The colony formation ability was detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. The protein expression levels were analyzed by Western blot. Results： Casticin significantly inhibited the growth of human cervical cancer HeLa cells in a dose- and time-dependent manner, and the IC50 was 2.82 μg/mL. The colony-forming rate was reduced drastically compared with control group （P 〈 0.05）. The cells were markedly arrested at G2/M phase after the treatment of Casticin for 48 h. Western blot showed that the expression of p21 protein was up-regulated and protein level of Cyctin B1 was depressed by Casticin in a concentration dependent manner. Conclusion： Casticin could inhibit the cell growth and lead to cell arrest in human cervical cancer HeLa cells, and the down-regulation of Cyclin B1 protein expression and activation of p21 protein might contribute to Casticin induced cell arrest in human cervical cancer HeLa cells.

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.

Effect of Coriolus Versicolor Polysaccharide-B on the Biological Characteristics of Human Esophageal Carcinoma Cell Line Eca109

Objective To investigate the effect of Coriolus versicolor polysaccharide-B （CVPs-B） on the biological characteristics of human esophageal carcinoma cell line Ecal09 in vitro. Methods The cells of experimental group （EG） were cultured in DMEM with 10% FCS and 150μg/mL CVPs-B, the cells of control group （CG） were cultured in DMEM with 10% FCS without CVPs-B. MTT reduction assay was performed to detect the effect of CVPs-B on the proliferation of Ecal09 cells after the compound was administrated in varying concentrations. The living conditions of the Ecal09 cells were determined using trypan blue exclusion. Then, cell growth curves were drawn. Flow cytometry was performed to detect the effect of CVPs-B on the apoptosis and cell cycle of Ecal09. Results In comparison with the CG, a marked decrease in the proliferation of Eca09 cells was observed in the EG, after incubation with CVPs-B. The survival rate of Eca09 cells decreased as the time of CVPs-B incubation prolonged. Comparing the cell cycles and apoptotic rates between the two groups, the proportions of cells in the G0/G1, S, and G2/M phases in the EG were found to be （68.4±3.7）%, （13.9±2.1）%, and （17.7±1.4）%, respectively, after 24 h incubation with CVPs-B. The cells had an apoptotic rate of （9.7±0.7）%. On the other hand, the proportions of the G0/G1, S, and G2/M cells of the CG were found to be （53.9±3.6）%, （26.6±2.8）%, and （19.5±2.3）%, respectively, with an apoptotic rate of （5.7±1.4）%. In comparison with the CG cells, significant cell growth in the G0/G1 phase was observed in the EG （P〈0.05）. Furthermore, a significant decrease in the number of cells in the S phase was observed （P〈0.05） in the EG. Conclusions CVPs-B can inhibit proliferation and enhance apoptosis of Ecal09 cells and may be useful in the treatment of esophageal carcinoma.

The δ^15N response and nitrate assimilation of Orychophragmus violaceus and Brassica napus plantlets in vitro during the multiplication stage cultured under different nitrate concentrations

Natural nitrogen isotope composition(δ^(15)N) is an indicator of nitrogen sources and is useful in the investigation of nitrogen cycling in organisms and ecosystems. δ^(15)N is also used to study assimilation of inorganic nitrogen. However, the foliar δ^(15)N of intact plants, which is a consequence of nitrate assimilation occurring in the roots and shoots, is not suited for studying nitrate assimilation in cases where nitrate is the sole nitrogen source. In this study, Orychophragmus violaceus(Ov) and Brassica napus(Bn) plantlets, in which nitrate assimilation occurred in the leaves, were used to study the relationship between foliar δ^(15)N and nitrate assimilation.The plantlets were grown in vitro in culture media with different nitrate concentrations, and no root formation occurred for the plantlets during the multiplication stage.Nitrogen isotope fractionation occurred in both the Ov and the Bn plantlets under all treatments. Furthermore, the foliar nitrogen content of both the Ov and Bn plantlets increased with increasing nitrate concentration. Foliar nitrogen isotope fractionation was negatively correlated with foliar nitrogen content for both the Ov and Bn plantlets. Our results suggest that the foliar nitrogen isotope fractionation value could be employed to evaluate nitrate assimilation ability and leaf nitrate reductase activity.Moreover, high external nitrate concentrations couldcontribute to improved foliar nitrogen content and enhanced nitrate assimilation ability.

The inhibiting effects of Laggera alata flavone on human ovarian cancer HO-8910 cells proliferation and its mechanism in vitro

The purpose of this study was to investigate the effect of Laggera alata flavonen （LAF） on the inhibit- ing effect of human ovarian cancer HO-8910 cells proliferation and its possible mechanism in vitro. Methods： Human ovarian cancer HO-8910 cells were cultured in vitro. Inhibitory effect of LAF on the viability of HO-8910 cells was evaluated by the MTT assay. Apoptotic effect of different concentrations of LAF on HO-8910 cells was assessed by AO/EB staining and FCM with propidium iodide （PI） staining. Expression of proteins related to apoptosis was analyzed by Western blot. Results： LAF significantly inhibited the viability of HO-8910 cells proliferation in a dose-dependent and time-dependent manner, there were statistical significance compared with NS group （P 〈 0.05）, and the ICso was 4.28 pg/mL for 48 h. The cells treated with LAF showed typical morphological change and apoptotic rate increased by FCM in a dose-dependent, and there was notable dif- ference compared with NS group （P 〈 0.05）. Western blot showed that expression of Fas, caspase-8, tBid and Cyto-c proteins were up-regulated after treatment with LAF for 48 h in a concentration dependent. Conclusion： LAF could inhibit HO-8910 cells proliferation and induce apoptosis, which may be through the pathway of death receptor in vitro.

Measuring Telomere Length in Proliferating Cells by Flow-FISH Method

The purpose of present work is a measurement of telomere length dynamic in proliferating cells in vitro by modified flow-FISH method. This method is a combination of two modifications： telomere length measurement in differentiated cells by surface antigen and analysis of cells divisions＇ number by vital dye dilution. Lymphocytes were activated by anti-CD3 Abs with IL-2 presents and grown in vitro for 7 days. Cells division＇s number was measured by dilution of CFSE vital dye which cells were stained previously activation. For telomere length measurement we used flow-FISH method with Cy3 labeled telomere PNH probe. Using this method we evaluated the dynamic of telomere length in CD4＋ and CD8＋ T-cells after 7 days culturing in vitro and revealed the difference in telomere lengthening and shortening versus division rounds in cell subsets. In CD8＋ cells telomeres start lengthen on a second division with the maximum on 4th division round becoming more that 20% longer compared with undividing cells. In CD4＋ cells telomeres did not have any length peculiarities through all division rounds demonstrating different telomere regulation in subsets. This probably occurs due to the higher level ofhTERT protein expression in CD8＋ than CD4＋ cells do.

In vitro incubation of cytokine-induced killer cells from patients with and without hepatitis B virus and a cell subset analysis

Objective The aim of the study was to explore the difference between immune cell subsets during the incubation of cytokine-induced kill cells （CIKs） from patients with and without hepatitis B virus （HBV）. Methods Peripheral blood samples were extracted from 50 tumor patients, and were divided into two groups according to the presence or absence of HBV. The proliferation rate and activity of CIK cells were examined based on counts on days 1, 5, 7, 9, 11, 13, and 15 of culture. Additionally, the CD3＋, CD4＋, CD8＋, CD3＋CD8＋, C＋）3＋CD4＋, and CD3＋CD56＋ T cell populations were analyzed by flow cytometry on days 5, 7, 10, 13, and 15 of culture. Results Proliferation over a 15-day period was higher in the HBV-positive group than in the negative group （280-fold vs. 180-fold increase, respectively）, but there was no significant difference between the two groups at each time point. The frequencies of CD3＋, CD8＋ T, CD3＋CD8＋, and CD3＋CD56＋T cells increased over time, while those of CD4＋ and CD3＋CD4＋ T cells decreased over time, and these changes were greater in the positive group than in the negative group. The differences in CD8＋ T cells and CD3＋CD4＋ T cells between the two groups were significant （P 〈 0.05）. Conclusion The proliferative capacity of CIK cells was higher for patients in the HBV-positive group than those in the HBV-negative group, and immune cell subsets were more favorable in the HBV-positive group than the neaative arouD.

左旋氨氯地平对缺氧心肌细胞钙超负荷的作用

目的观察左旋氨氯地平对缺氧心肌细胞钙离子超负荷的保护性作用。方法体外增养心肌细胞并建立心肌细胞缺氧模型,采用Fura2/am荧光探针在荧光分光光度计下直接测定各组心肌细胞内的游离钙离子浓度,通过RT-PCR方法分析细胞质膜上钙离子ATP酶的表达,并同时用WesternBlot方法测定肌浆网上钙泵的量。结果在左旋氨氯地平干预下缺氧心肌细胞的游离钙离子浓度显著降低(359.06±75.00nmol/L),同时增强了缺氧条件下的细胞质膜钙离子ATP酶的表达及肌浆网上钙泵的含量。结论左旋氨氯地平可以通过提高钙泵的表达来增强心肌细胞在缺氧条件下对钙超负荷的抵抗能力。