大鼠胰岛分离、培养条件的优化及miR-126表达的检测方法比较

Optimization of SD Rat Islet Cell Isolation,Culturing and the Comparison of Methods Detecting miR-126

目的:优化SD大鼠胰岛细胞的分离、纯化和培养方法与条件,为研究miR-126在Ⅱ型糖尿病中的作用机制提供活性与功能良好的胰岛细胞及miR-126表达的检测方法。方法:水合氯醛腹腔注射麻醉SD大鼠,采用8 mL胶原酶Ⅴ(含DNaseⅠ100 U)逆行注射、原位消化后Hitopaque-1077梯度离心分离纯化SD大鼠胰岛细胞,从培养后的胰岛细胞中提取总RNA,分别用加尾法和茎环法进行miR-126的反转录,实时定量PCR(qPCR)检测miR-126的表达量。结果:用该方法可从每只SD大鼠中分离、纯化得到胰岛细胞372±45个,胰岛细胞纯度>90%,胰岛细胞存活率>95%;用加尾法和茎环法qPCR检测miR-126的Cp值分别为34.56±2.56和32.47±2.01。结论:胶原酶Ⅴ(含DNaseⅠ100 U)逆行注射、原位消化可有效避免因消化时胶状物质的产生而导致的胰岛细胞分离失败,Hitopque-1077梯度离心分离方法具有操作简单、便捷、成功率高等特点,可得到活性与功能较好的胰岛细胞;与加尾法相比,茎环法能够更灵敏地检测胰岛细胞miR-126的表达量。

Objective： In order to provide active and functional islet cells for research on the role and mechanism of miR-126 in type Ⅱ diabetes disease, we explored the optimal isolation method and culture condition of SD rat islet cells and compared the detection methods for miR-126. Methods： SD rats were anesthetized through intraperitoneal injection with chloraldurat, then 8 mL of collagen V containing 100 U of DNase I was administrated through retroperfusion. After in situ digestion, SD rat islet cells were isolated and purified with gradient centrifugation in Hitopaque-1077. Total RNA of cultured islet cells was extracted, and the amount of miR-126 was measured with methods of plus poly（A） tail or stem-loop. Results： By using the isolation method described as above, the average number of islet cells isolated from a SD rat could reach 372±45. The purity of isolated islet cells was larger than 90% and their viability were over 90%. When detecting the expression of miR-126 by using quantitative PCR（qPCR）, the crossing point was 34.56±2.56 and 32.47±2.01 respectively when using method of plus poly（A） tail or stem-loop. Conclusion： Retroperfusion with collagen V plus DNase I can effectively avoid colloidal substances produced when digesting the islet cells which leading to the failure of isolation. Gradient centrifugation in Hitopaque-1077 is an effective method to isolate and purify SD rat islet cells, which is simple, convenient and with high success rate. Quantitative method of stem-loop is more sensitive than plus poly（A） tail in detection of miR-126 expression in islet cells.