MTT法检测黄芪和丹参对人乳腺干细胞体外增殖的影响

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—0912007—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1．0mm’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1×10^8L^-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25，50，100，200mI几4个亚剂量；丹参注射液组分为12．5，25，50，100mL/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72h，加入5g/L MTT溶液20μL，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50mL/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200mL/L时产生抑制作用（P〈0．05）。丹参注射液剂量在12．5～100mL/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/

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

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 mm^3. 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 cuiture.②Proliferation of the mammary cells： With 50 mL/L as a boundary, RA could promote