摘要
背景:一些实验证明多糖在感染、炎症、细胞间黏附和信号传导、免疫识别、细胞增殖分化及维持细胞正常结构和功能生理、病理等方面均起重要作用。但植物多糖对胃肠道黏膜的保护作用有待进一步研究。目的:观察不同浓度的四君子汤总多糖对大鼠小肠上皮株IEC-6细胞增殖的影响。设计:观察对比实验。单位:广东省中医院中心实验室。材料:①细胞株:正常大鼠小肠上皮细胞株IEC-6(ATCCCatalogNo.RL-1592)由美国ATCC(AmericanTypeCultureCollection)购得,主要来源于小肠隐窝细胞。②试剂和药品:DMEM培养基,牛胰岛素,庆大霉素,胎牛血清,DPBS均为GIBCO公司产品;细胞增殖试剂盒(MTT)为Roche公司产品;消炎痛为Sigma公司产品。四君子汤总多糖按药典处方比例取党参,白术,茯苓和甘草适量,加水浸泡后,煮沸提取4h,重复1次,合并提取液,减压浓缩,高速离心除去不溶物,提取液置玻璃纸中逆向流水透析2h,再于提取液中加乙醇至浓度约80%,保存过夜后分取沉淀,合并所有沉淀,采用Sevag法除去粗多糖中的蛋白,冻干后得到的总多糖。方法:①将IEC-6细胞株于干冰中取出,迅速放入37℃水浴中解冻后,加入已装有DMEM培养液的T-150培养瓶后放入CO2培养箱中,于37℃,饱和湿度,体积分数为0.05的CO2环境下培养。细胞隔天换液1次,每5～7天进行传代,传代比例为1∶7。第15～20代细胞用于实验。②IEC-6细胞以1×104/孔的浓度接种于96孔细胞培养板上,接种后6h,各培养孔内依次加入浓度为50,100,200mg/L四君子汤总多糖,即为四君子汤总多糖3个剂量组。每天定时取出1板,按试剂盒说明书以MTT法进行细胞增殖检测。以未加任何干预措施的IEC-细胞作为正常对照组,在相应时间点以MTT法进行细胞增殖检测。③将IEC-6细胞按1×104/孔的浓度接种于96孔细胞培养板中,接种后次日更换以无血清培养液DMEM,24小时后,各培养板中分别加入消炎痛以及四君子汤总多糖,其中,消炎痛使用浓度为40mmol/L,即为消炎痛组。四君子汤总多糖使用3个剂量分别为50,100,200mg/L,即为四君子汤总多糖50,100,200mg/L组。药物作用20h后,按试剂盒使用说明书进行MTT法细胞增殖检测。以未加任何干预措施的IEC-细胞作为正常对照组,在各相应时间点以MTT法进行细胞增殖检测。主要观察指标:MTT法测定四君子汤总多糖不同作用时间对IEC-6细胞生长的影响结果;MTT法测定四君子汤总多糖对消炎痛所致的IEC-6细胞生长抑制的影响结果。结果:四君子汤总多糖不同剂量于不同的培养时间对IEC-6细胞的生长均有促进作用;IEC-6细胞在给予消炎痛40mmol/L作用24h后,细胞增殖的吸光度明显低于正常对照组(0.17±0.02,0.31±0.03;P<0.01)。当四君子汤总多糖使用浓度为100mg/L时,IEC-6细胞的吸光度明显高于消炎痛组(0.25±0.04,0.17±0.02;P<0.01),虽然未达到正常IEC-6细胞吸光度的水平,但与正常组无明显差异(P>0.05)。结论:四君子汤总糖具有促进IEC-6细胞增殖的作用,它可能通过对小肠上皮细胞增殖的影响而发挥对肠道黏膜吸收和免疫功能的调整作用。
BACKGROUND： In a series of recent studies it was demonstrated that polysaccharides play important roles in many physiologic and pathologic processions, such as infection, inflammation, inter-cell adherence and signal conduction, immune identification, cell proliferation and differentiation, as well as maintenance of cell structure and function. But the protective effect of plant polysaccharides on gastrointestinal mucosa needs further research. OBJECTIVE： To observe the effect of the total polysaccharides of Sijunzi Decoction （SJZD） （TPSJ） in different concentrations on the proliferation of rat intestinal epithelial cell line IEC-6. DESIGN： Observational controlled trial. SETTING： Central Laboratory, Guangdong Hospital of Traditional Chinese Medicine. MATERIALS： ①Cell line： The IEC-6 of normal rats （Catalog No. RL- 1592） was purchased from American Type Culture Collection （ATCC）. IEC- 6 cells were originated mainly from intestinal crypt cells. ②Reagents and drugs： DMEM medium, bovine insulin, gentamicin, fetal bovine serum （FBS） and DPBS were purchased from GIBCO Ltd. Cell proliferation kit （MTT） was purchased from Roche Ltd. lndomethaein was purchased from Sigma Company. SJZD was composed of Dangshen （Codonopsis pilosula）, Baizhu （Atractylodes macrocephala）, Fuling （Poria cocos） and Gancao （Glycyrrhizae uralensis）, and these four drugs were in same ratio as Pharmacopoeia. The four herbs were boiled in water, extracted twice for 8 hours. Extract was combined, decompressed, concentrated, centrifugated with high speed to take out insoluble substance, put in glass paper to receive reverse lotic water dialysis for 2 hours. The final decoction was concentrated by heating followed by extraction with 80% ethanol. After overnight precipitation at room temperature and combination of sedimen, the total polysaccharide was obtained by deproteinating with the Sevag method. METHODS： ①The IEC-6 cell line was maintained in T-150 flasks with DMEM culture solution, and then put in CO2 incubator at 37℃, at saturated humidity, cultured at 0.05 volume fraction CO2, after being taken out from dry ice and defrosted rapidly in water-bath at 37 ℃. Flasks were incubated at 37 ℃ in 5% CO2. Stock cells were subcuhured at a dilution of 1：7 every 5-7 days and the medium was changed once every 2 days. The cells in passage 15-20 were used for testing. ②IEC-6 cells were inoculated at a density of 1×10^4 cells/well in 96-well plates. Cultured were supplemented with TPSJ in a final concentrations ranging from 50, 100 and 200 mg/L after 6 hours, which was 3 TPSJ groups. One plate would be taken out for the examination of cell proliferation using MTT assay everyday. The cells that not administrated by any intervention were used as normal control group and cell proliferation was assayed using MTT at corresponding time points. ③IEC-6 cells were inoculated at a density of 1×10^4 cells/well in 96-well plates, and then cultured in the DMEM supplemented with no serum from the following day for 24 hours. For the examination of mucosal restitution, indomethacin at concentration of 40 mmol/L was employed to induce IEC-6 cells injured, which was indomethacin group. The three concentration of TPSJ was 50, 100 and 200 mg/L, respectively, which was 50,100,200 mg/L TPSJ groups. After drug action for 20 hours, the proliferation of cells was measured using MTT according to the manufacturer＇s instructions. used in the normal control group IEC-cells without any intervention were Cell proliferation was determined with MTT method at corresponding time points. MAIN OUTCOME MEASURES： MTT assay was used to examine the effects of TPSJ on IEC-6 cell proliferation in different times. MTr assay was used to detect the effect of TPSJ on IEC-6 cell proliferation inhibited by indomethacin. RESULTS： TPSJ could accelerate IEC-6 cells growth at different doses and in different time. After the cells were treated by 40 mmol/L indomethacin for 24 hours, the absorbance （A） of IEC-6 cells apparently declined compared with that in the normal control group （0.17±0.02, 0.31±0.03; P 〈 0.01）. The A of IEC-6 cells treated by TPSJ in 100 mg/L group was apparently higher compared with indomechacin group （0.25±0.04, 0.17±0.02; P 〈 0.01）. The A of IEC-6 cells treated by TPSJ did not restored to the normal level, but there was no insignificant difference compared with normal group （P 〉 0.05）. CONCLUSION： TPSJ can accelerate the proliferation of IEC-6 cells. TPSJ can exert regulatory function both in intestinal mucosa absorption and immunity by affecting intestinal epithelial cells.
出处
《中国临床康复》
CSCD
北大核心
2006年第35期175-177,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30371769)~~
