课题背景：课题以人非家族性乳腺癌癌旁组织中分离的成体乳腺干细胞为对象，经原代培养，免疫磁珠分离，单细胞培养，连续转染猿病毒40大T抗原片段后能够连续传代约2～4个月，经免疫组化和印记杂交鉴定细胞表达ESA抗原及K19，而不表达sialomucin，在三维培养中表现出形成腺泡结构的功能。采用反转录PCR及免疫印记杂交方法，观察干细胞及癌组织中乳腺癌相关基因的表达，及染色体部分位点的基因组不稳定性。现阶段成果显示乳腺干细胞无相应癌基因表达、抑癌基因变异或缺式，可以证明乳腺干细胞不具有恶性变的分子基础。

同行评价：各种干细胞的增殖与分化调控是干细胞研究的重要课题，乳腺干细胞有分化成乳腺各种上皮细胞及形成乳腺腺体结构的能力。本实验探讨黄芪和丹参对体外培养的乳腺干细胞增殖的影响，发现合适浓度的单药可以促进乳腺干细胞增殖，且两种药物联合使用产生协同效果，对乳腺干细胞的最终应用有一定积极意义。

偏倚或不足：①实验检测技术较单一，仅采取MTT比色法分析不同剂量黄芪和丹参对体外分离培养的乳腺成体干细胞增殖的影响，缺乏更深层次的分析。②实验重复性未得到论证。③药物中起作用的主要成分、联合协同作用的机制尚不清楚。

摘要
目的：文献报道黄芪和丹参均可诱导间质干细胞向神经细胞分化。实验以乳腺癌患者乳腺再生为最终目的，通过MTT比色法分析不同剂量黄芪和丹参对体外分离培养的乳腺成体干细胞增殖的影响。
方法：实验于2006-09/2007-09在吉林大学公共卫生学院放射生物实验室完成。①对象：女性非家族性乳腺癌癌旁病理证实为正常的乳腺组织取自吉林大学第一医院乳腺外科，共24例，患者年龄29～45岁，对治疗及实验均签署知情同意书。②实验方法：取切除的正常乳腺组织，去除血管及脂肪，剪碎至1.0 mm3块，组织块消化培养传代后应用免疫磁珠分离收集MUC-、ESA+标记的细胞，即乳腺成体干细胞，按1×108 L-1密度接种于96孔培养板，无血清培养48h后更换培养基，设立4组：空白对照组不加任何药物；黄芪注射液组分为25，50，100，200 mL/L 4个亚剂量；丹参注射液组分为12.5，25，50，100 mL/L 4个亚剂量；黄芪+丹参复合注射液组分为（25+12.5）,(50+25),（100+50）,（200+100）mL/L 4个亚剂量。③实验评估：各组加入对应药物后培养72 h，加入5 g/L MTT溶液20 μL，再加入二甲基亚砜振荡溶解，在酶联免疫检测仪上测定各孔的吸光度值，分析乳腺干细胞的增殖情况。
结果：①乳腺细胞形态观察：乳腺组织原代培养后形成腺上皮细胞和肌上皮细胞。②乳腺干细胞增殖检测：黄芪注射液以 50 mL/L为界，剂量越小对乳腺成体干细胞的促增殖作用越强（P < 0.01），达200 mL/L时产生抑制作用（P < 0.05）。丹参注射液剂量在12.5~100 mL/L范围内变动时，均能够促进乳腺成体干细胞的增殖（P > 0.05）。黄芪与丹参复合注射液以（100+ 50）mL/L为界，复合剂量越小对乳腺成体干细胞的促增殖作用越强（P < 0.01），且复合用药效果好于单独用药，当剂量达到（200+100）mL/L时产生抑制作用（P < 0.05）。
结论：丹参和小剂量黄芪均可提高体外分离培养的乳腺干细胞增殖能力，且两药低剂量联合使用促增殖作用更为明显。
关键词：乳腺成体干细胞；黄芪；丹参；细胞培养

王蕾，张科伟，赵大伟，付士波，刘国津，石爱平. MTT法检测黄芪和丹参对人乳腺干细胞体外增殖的影响[J].中国组织工程研究与临床康复，2008，12(8):1418-1421 [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1418(ps).pdf]

Effects of radix astragali and salvia miltiorrhiza bunge on the proliferation of human mammary stem cells detected with MTT chromatometry

Abstract

AIM：It is reported that radix astragali (RA) and salvia miltiorrhiza bunge (SMB) can induce mesenchymal stem cells differentiate into nerve cells. This study analyzed the effects of RA and SMB on the proliferation of the adult mammary stem cells cultured in breast cancer patients by means of MTT chromatometry.

METHODS: The experiment was done in the Radio-biochemical Laboratory, Public Health College of Jilin University during September 2006 to September 2007.①Twenty-four female patients who haven't familial breast cancer, aged 29-45 years, were adopted in the study. The glandular tissue obtained from Department of Mammary Surgery in the First Hospital of Jilin University, all the patients had signed informed consent.②Vessel and fat were removed from the normal mammary gland tissues which had been excised, and then crushed into pieces of 1.0 mm3. After digestion, cultivation and purification by immunomagnetic sorting, the mammary adult stem cells labeled with MUC- and ESA+ were harvested and inoculated into a 96-well culture plate. Serum-free culture medium was changed 48 hours later, and divided into four groups: blank control group was untreated; RA group was added with different concentrations of RA (25, 50, 100, 200 mL/L); SMB group was added with different concentrations of SMB (12.5, 25, 50, 100 mL/L); Combined group was added with RA and SMB [(25+12.5), (50+25), (100+50), (200+100) mL/L].③After the cells were cultured for 72 hours, 5 g/L MTT solution (20 μL) was added, then dimethyl sulfoxide was used for vibration and dissolvement. Optical density of each hole was detected by using enzyme-linked immunosorbent assay to analyze different concentrations of RA and SMB on the proliferation of human mammary stem cells.

RESULTS: ①Morphous of the mammary cells: The tissues formed glandular epithelial cells and myoepithelial cells after the primary culture.②Proliferation of the mammary cells: With 50 mL/L as a boundary, RA could promote the proliferation of human mammary stem cells, and the smaller the dosis was, the better the effect was (P < 0.01), 200 mL/L brought about inhibition (P < 0.05). SMB could promote the proliferation of human mammary stem cells at the range of 12.5-100 mL/L (P > 0.05). Starting from (100+ 50) mL/L, the combination of RA and SMB could promote the proliferation of human mammary stem cells, the smaller dosis indicated the better effect (P < 0.01), and the combination was superior to the single use. (200+100) mL/L combination brought about inhibition (P < 0.05).

CONCLUSION: SMB and small dose of RA can promote the proliferation of human mammary stem cells. The effect is more obvious when RA and SMB combined at small dose.

Wang L, Zhang KW, Zhao DW, Fu SB, Liu GJ, Shi AP.Effects of radix astragali and salvia miltiorrhiza bunge on the proliferation of human mammary stem cells detected with MTT chromatometry.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1418-1421(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1418(ps).pdf]