目的：了解人乳腺癌细胞系MCF-7中的不同亚群细胞的体外增殖特性。方法：采用流式细胞技术从MCF-7细胞中分选出不同的细胞亚群，并测定它们在体外的增殖、克隆能力。结果：和CD44+CD24+细胞相比，MCF-7中的CD44+CD24-细胞具有更强的体外克隆能力，而两者的体外增殖能力相近。结论：MCF7中存在乳腺癌干细胞，而CD44+CD24+细胞可能是乳腺癌祖细胞。
【Abstract】 Objective To investigate the role of myosin l ight chain (Myl) in myogenesis in vitro. Methods The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb prol iferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the abil ity of prol iferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was indentified as the control group. Results The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P lt; 0.01), and the latter two did not show any significant difference (P gt; 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The prol iferation assay showed that the eMb prol iferated faster as compared with dMb and gMb (P lt; 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 ± 24.5)/ field, which was significantly larger than that of myotubes from dMb [(47.6 ± 15.5) / field ] and gMb [(39.8 ± 5.1) field ] (P lt; 0.01). There was not statistically significant difference between the latter two groups (P gt; 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P lt; 0.05), and the dose-dependent way was performed.The numbers of myotubes from dMb at 16 hours were (48.2 ± 7.1)/ well in the experimental group and (23.4 ± 4.9)/ well in the control group, and at 6 days were (40.6 ± 10.2)/ field in the experimental group and (63.1 ± 6.1)/ field in the control group.There was statistically significant difference between the experimental and control groups (P lt; 0.01). Conclusion Myl may play a role in myogenesis through the negative effect on the myoblast prol iferation.
Objective To investigate the culture method forepidermal stem cells in vitro. Methods The epidermis was separated from the dermis, and shaken for 10 min in 0.05% trypsin at 37℃ to dissociate into single cells. Epidermal stem cells were selected by rapid attachment to collagen Ⅳ for 10-15 min and cultured on collagen Ⅳ or 3T3 feeder layer. All the cells were grown in DMEM without calcium, supplemented with 10% chelexed fetalbovine serum, 10 μg/L epidermal growth factor, 0.05 mmol/L CaCl2 and 0.8 mg/L hydrocortisone. Cultures were observed for colony formation under a phase constrast microscope. The phenotypes of epidermal stem cells were detected by flow cytometry and immunocytochemistry staining. Results The cells selectedby rapid adherence to collagen Ⅳ formed large colonies at 7～8 days, expressedK19 antigen. The percentages of cells at the G0 and G1 phases of the cell cycle and the percentage of α6briCD71dim cells in the experimental groups were higher than those in the control group. It indiciated that there was a significant difference between the experimental groups and the control groups(P＜0.05). ConclusionThe humanepidermal stem cells can be selected by rapid attachment to collagen Ⅳ, and they can be expanded in culture if the appropriate conditions are maintained.
Objective To investigate the role of anti apoptosis gene bcl-2 in the survival of cultured uveal melanoma UM cells. Methods Antisense oligodeoxynucleotides (AS-ODN) bcl-2 were delivered with cationic lipid to primary cultured UM cells. The inhibitory effect of AS-ODN bcl-2 on proliferation of UM cells was examined by 3,-4,5 Dimethyliazol-2,5 diphenyl tetrazolium bromide (MTT) method. Using DNA ladder to determine if the UM cells had been apoptotic. Bcl-2 expression was detected by RT-PCR and Westernblot technics. Results The effect of AS-ODN bcl-2 in inhibiting the proliferation of cultured UM cells had opposite relation to dosage. It down regulated the mRNA and protein level of bcl-2 gene, and the sense ODN didn′t have this effect. Conclusion AS-ODN bcl-2 can down regulate bcl-2 expression, inhibits UM cells proliferation and induces apoptosis. (Chin J Ocul Fundus Dis, 2002, 18: 38-41)
目的 系统评价可作为将基础生命科学研究从实验室转化到人体研究和健康保健措施的工具。对动物研究的系统评价是否系统且不存在偏倚，目前尚不知。 方法 我们检索了MEDLINE、EMBASE、已知系统评价的参考文献（1996~2004）并联系相关专家，至少检索一个对公众开放的数据库全面收集基础科学文献的引文，均未设定语言限制。我们从中纳入那些在动物身上测量实验室参数或在活体动物身上给予治疗以评价疗效研究的系统评价，并将其与在实验室中研究人体或动物组织、细胞系统或器官标本以进一步了解疾病机理体外研究的系统评价进行比较。 结果 动物研究的系统评价通常缺少如定义供检验的假设（9/30，30％）；文献检索不设语言限制（8/30，26.6％）；评价发表偏倚（5/30，16.6％）、研究的真实性（15/30，50％）和异质性（10/30，33.3％）；及使用Meta分析用于定量合成（12/30，40％）等方法学特征。与体外研究的系统评价相比，动物研究的系统评价更多地限定了研究问题（96.6％ vs. 80％，P=0.04）、综合检索了多个数据库（60％ vs. 26.6％，P=0.01）、评价了研究质量（50％ vs. 20％，P=0.01）及探讨了研究异质性（33.3％ vs. 2.2％，P=0.001），因此更少出现偏倚。 结论 在各类系统评价中，方法学质量问题的出现频率似乎存在一定的梯度：虽然与人体临床试验的系统评价相比，整体动物研究的系统评价质量明显较差，但其仍优于体外研究的系统评价。在系统评价动物研究时，有必要更严格。
目的：通过体外研究探讨化疗剂量对乳腺癌治疗效果的影响。方法：通过手术取材和肿瘤组织原代细胞体外培养技术，对96例乳腺癌患者进行体外药物敏感性检测。结果:乳腺癌晚期患者比早期患者的耐药风险明显增加（P＜0.05），乳腺癌复发患者比原发患者具有更强的多药耐药能力（P＜0.01）；增加化疗药物剂量仍然可以提高大多数耐药患者的治疗效果（P＜0.01）。结论：大剂量化疗方案对晚期和复发性乳腺癌患者有帮助。
Objective To understand the effect of estradiol in different concentrations on proliferation of diverse mammary primary cells in vitro. Methods The primary cells of cancer tissue, the adjacent tissue to tumors and normal mammary tissue from patiens with breast cancer were obtained using collagenase digesting method. All the tissue samples were cultivated in vitro, and were given estradiol in different concentrations. The effect of estradiol on the proliferation of those primary cells was measured by MTT. Results Estradiol remarkedly promoted the proliferation of primary cells of cancer tissue and peritumor tissue in vitro, whose ER expression were positive. Whereas, the promotion effect of estradiol on the proliferation of normal mammary primary cells was relatively weak, and there was no correlation between the promotion effect with the expression of ER in cancer tissue. Conclusion The risks of occurrence and relapse of breast cancer would increase significantly when the concentration of estradiol is no less than 103 pmol/L in vivo.
Objective To observe the synergistic effect of metformin and anti-vascular endothelial growth factor (VEGF) in the treatment of diabetic retinopathy. Methods This study was composed of clinical data review and in vitro cell experiment. Ten patients (12 eyes) with diabetic macular edema treated with anti-VEGF drugs were included in the study. Patients were randomly divided into the VEGF group (anti-VEGF drug therapy) and the combined treatment group (anti-VEGF drug combined with metformin). The changes of visual acuity and central retinal thickness (CRT) were compared between the two groups. As far as the in vitro experiment was concerned, vascular endothelial cells were divided into the control group (normal cells), the VEGF group (50 ng/ml VEGF), the anti-VEGF group (50 ng/ml VEGF+2.5 μg/ml of conbercept), and the combined group (50 ng/ml VEGF +2.5 μg/ml of conbercept +2.0 mmol/L of metformin). And then MTT cell viability assay, scratch assay and real-time quantitative polymerase chain reaction assay were performed to analyze the cell viability, cell migration and mRNA level of VEGFR2, protein kinase C (PKC)-α and PKC-β successively. ResultsReview of clinical trial shows that the CRT recovery rates in the combined treatment group were much higher than that in the VEGF group at 3 month after the operation, while the difference was statistically significant (t=−2.462, P＜0.05). In vitro cell experiment results showed that VEGF induction upregulated the viability and mobility of vascular endothelial cells obviously compared with control group, at the same time, the use of anti VEGF drugs can effectively reverse the trend, in contrast, combination of metformin and anti-VEGF showed a more superior effect to some extent (P＜0.05). In the VEGF group, the mRNA expression of VEGFR2, PKC-αand PKC-β were significantly increased compared with the control group (P＜0.01); while the mRNA expression of VEGFR2, PKC-αand PKC-β in the combination group decreased significantly compared with the VEGF group and the control group (P＜0.05). However, in the anti-VEGF group, the mRNA expression of VEGFR2, PKC-αand PKC-β were decreased, but has failed to reach the level of statistical learn the difference. ConclusionsThe combination of metformin and anti-VEGF drugs can reduce the CRT of diabetic retinopathy patients and inhibit the proliferation and migration of retinal vascular endothelial cells which induced by VEGF. The synergistic mechanism may be related to the inhibitory effect of metformin on the expression of VEGFR and PKC.