不同胶原酶消化对大鼠胰岛的纯化和获得率的影响

Effects of different collagenase digestion on yield rate of purified islets in rats

目的比较不同胶原酶消化大鼠胰腺的效果，选择消化效果较好的胶原酶。方法采用随机数字表法将SD大鼠分为2组，胶原酶P组大鼠胰腺使用1mg／ml的胶原酶P液进行消化，V型胶原酶组大鼠胰腺使用1mg／ml的V型胶原酶进行消化。胰腺消化后，对获得的两组大鼠胰岛进行纯化、培养。采用双硫腙染色于倒置显微镜下计数纯化前后获得的两组全部胰岛数量，并计算胰岛当量（IEQ）；采用吖啶橙和碘丙啶双色荧光染色，于荧光显微镜下计数纯化后胰岛的活率；两组大鼠胰岛经纯化、培养2d后进行胰岛素释放试验，观察两组胰岛的功能。结果纯化前，两组间每个大鼠胰腺获得的胰岛数量的差异无统计学意义（P〉0．05）；但两组间IEQ的差异有统计学意义（P〈0．05）；经纯化后，V型胶原酶组和胶原酶P组每个大鼠胰腺获得的胰岛数量分别为485±113和643±82，IEQ分别为674±157和989±126，两组间胰岛数量和IEQ的比较，差异均有统计学意义（P〈0．05）。V型胶原酶组和胶原酶P组大鼠胰岛经纯化后，其活率分别为（96．13±1．13）％和（96．38±0．92）％，两组比较，差异无统计学意义（P〉0．05）。胰岛素释放试验结果显示，在低糖或高糖刺激下，两组间大鼠胰岛功能的差异均无统计学意义（P〉0．05）。结论两种胶原酶均适用于大鼠胰腺的消化，但胶原酶P消化效果较V型胶原酶稳定，且胰岛获得率也较高。

Objective To compare the yield rate of rats islets between different collagenase digestion groups. Methods The SD rats were randomly divided into two groups as following by using random digits table： collagenase P group （pancreas digested by 1 mg/ml collagenase P） and type V collagenase group （pancreas digested by 1 mg/ml type V collagenase）. After pancreas digestion, rat islet cells in two groups were culture, purified and stained with DTZ. The mean islet number and islet equivalent （IEQ） before and after purification were measured under an inverted microscope The viability of purified islets was assessed by fluorescence staining of aridine orange （AO） and propidium iodide （PI） under the fluorescence microscopy. After purification and culture for two days, islets function was evaluated by insulin releasing tests in the two groups. Results Before purification, there was no significant difference in the islets number obtained from the pancreas between two groups （P〉0. 05）, but there was significant difference in the IEQ （P〈0. 05）. After purification, the islets number in type Vcollagenase group and collagenase P group was （485 ± 113）/ pancrease and （643 ± 82）/pancrease, and IEQ was （674 ± 157）/pancreas and （989 ± 126）/pancreas, respectively （P〈0. 05）. Islet viability in type V collagenase group and collagenase P group was （96. 13 ± 1.13） % and （96. 38 ± 0. 92） % respectively （P〉0. 05）. The results of insulin releasing tests revealed there was no significant difference in islet function stimulated by hypoglycemia and hyperglycemia between two groups （P〉0. 05）. Conclusion Two types of collagenase are suitable for the islets digestion in rats. The stability of digestion and yield rate of purified islets in collagenase P group are higher than in type V collagenase group.