小鼠胰岛提取方法改良的实验研究

曾文; 刘坤莹; 林楚文; 林硕; 彭航娅; 李海成; 曾龙驿

doi:10.3969/j.issn.1674-7445.2020.05.007

小鼠胰岛提取方法改良的实验研究

doi: 10.3969/j.issn.1674-7445.2020.05.007

曾文^{1, 2,},
刘坤莹^{1, 2},
林楚文^{1, 2},
林硕¹,
彭航娅^{1, 2},
李海成^{1, 2},
曾龙驿^{1, 2, ,}

1.
510630 广州, 中山大学附属第三医院内分泌与代谢病科
2.
广东省糖尿病防治重点实验室

基金项目:

国家自然科学基金面上项目 51873071

广东省自然科学基金重点项目 2018B030311012

详细信息

作者简介:
曾文, 女, 1988年生, 博士, 住院医师, 研究方向为糖尿病, Email：543149311@qq.com

通讯作者:
曾龙驿, 男, 1964年生, 博士, 教授, 研究方向为糖尿病, Email：zengly@mail.sysu.edu.cn

中图分类号: R617, R587.1
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- 被引次数: 0
出版历程
- 收稿日期: 2020-05-17
- 网络出版日期: 2021-01-19
- 刊出日期: 2020-09-15

Experimental study of a modified extraction method of mouse islets

Zeng Wen^{1, 2
,},
Liu Kunying^{1, 2},
Lin Chuwen^{1, 2},
Lin Shuo¹,
Peng Hangya^{1, 2},
Li Haicheng^{1, 2},
Zeng Longyi^{1, 2
, ,}

1.
Department of Endocrinology and Metabolic Diseases, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

More Information

Corresponding author: Zeng Longyi, Email:zengly@mail.sysu.edu.cn

摘要

摘要: 目的探讨小鼠胰岛提取方法的改良及效果。方法根据胰岛提取方法不同, 将小鼠随机分为胆总管穿刺组和胆总管穿刺联合胰腺原位注射组(联合注射组), 每组各100只。胰岛提取的改良方法采用胆总管穿刺联合胰腺原位注射法灌注胰腺, 体视显微镜下挑选并纯化胰岛。鉴定胰岛的形态和纯化情况, 统计两组的胰岛产量及胰岛提取成功率。分析胰岛在体外培养1周内的存活情况, 并评估体外培养24 h和4 d后胰岛的胰岛素分泌功能。结果与胆总管穿刺组比较, 联合注射组提取胰岛的产量显著增高(P < 0.001)。两组胰岛提取成功率均为83%, 差异无统计学意义(P > 0.05)。胆总管穿刺联合胰腺原位注射法所提取的胰岛形态完好、纯度高、活性好。新鲜分离的胰岛体外培养24 h后胰岛存活率接近100%;体外培养1~5 d, 胰岛细胞存活情况良好; 体外培养6 d开始, 胰岛出现中心性死亡。体外培养24 h和4 d后, 给予高葡萄糖刺激胰岛后, 小鼠胰岛功能均正常。结论胆总管穿刺联合胰腺原位注射法可以提高胰岛的产量, 且获取的胰岛细胞活性和功能良好。
- 胰岛 /
- 胰岛移植 /
- 胆总管穿刺 /
- 原位注射 /
- 胰岛素 /
- 胰高血糖素 /
- 胶原酶P /
- 双硫腙
Abstract: Objective To investigate the improvement and effect of the method of islet extraction in mice. Methods According to different islet extraction methods, all mice were randomly divided into the common bile duct puncture group (n=100) and common bile duct puncture combined with in situ pancreatic injection group (combined injection group, n=100). Common bile duct puncture combined with in situ pancreatic injection was utilized as the modified method. The islets were selected and purified under stereomicroscope. The morphology and purification of islets were identified. The islet yield and success rate of islet extraction were statistically compared between two groups. The survival of islets after 1 week culture in vitro was analyzed, and the insulin secretion function of islets after 24 h and 4 d culture in vitro was evaluated. Results Compared with the common bile duct puncture group, the islet yield in the combined injection group was significantly increased (P < 0.001). The success rate of islet extraction in both groups was 83% with no statistical significance (P > 0.05). The islets extracted by common bile duct puncture combined with in situ pancreatic injection had intact morphology, high purity and high activity. The survival rate of newly isolated islets was nearly 100% after 24 h culture in vitro. After 1~5 d culture in vitro, the islet cells survived well. After 6 d culture in vitro, the islets showed central death. After culture in vitro for 24 h and 4 d, the islet function of the mice was normal after high glucose stimulation. Conclusions Common bile duct puncture combined with in situ pancreatic injection can increase the islet yield, and the obtained islet cells have high activity and proper function.
- Islet /
- Islet transplantation /
- Common bile duct puncture /
- In situ injection /
- Insulin /
- Glucagon /
- Collagenase P /
- Dithizone

HTML全文

图 1 胆总管穿刺联合胰腺原位注射法的操作过程

注：A图为胆总管穿刺成功后，胰腺充盈；B图为联合胰腺原位注射胰头；C、D图为联合胰腺原位注射胰体；E图为联合胰腺原位注射胰尾；F图为联合胰腺原位注射后胰腺充分膨胀。

Figure 1. Operation procedure of common bile duct puncture combined with in situ injection of pancreas

下载: 全尺寸图片幻灯片

图 2 分离纯化后的小鼠胰岛形态

注：A图为DTZ染色提取的小鼠原代胰岛(DTZ, ×100);B图为DTZ染色纯化后的小鼠原代胰岛(DTZ, ×100);C~E图为纯化后的小鼠胰岛的胰岛素免疫荧光染色图(免疫荧光, ×200)。

Figure 2. Morphology of mouse islets after separation and purification A

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图 3 小鼠胰岛体外培养后的胰岛素和胰高血糖素双免疫荧光染色图(免疫荧光, ×400)

注：绿色为胰岛素, 红色为胰高血糖素。

Figure 3. Double immunofluorescence staining images of insulin and glucagon of mouse islet after in vitro culture A

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图 4 小鼠胰岛体外培养后的活性评估(免疫荧光, ×400)

注：A、B、C、D、E、F、G、H图分别为体外培养0 h、24 h、2 d、3 d、4 d、5 d、6 d、7 d后, 通过Hoechst 33342-PI荧光染色的原代胰岛细胞。

Figure 4. Activity evaluation of mouse islets after in vitro culture

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图 5 小鼠胰岛体外培养后的胰岛素功能评估

注：A图为小鼠胰岛体外培养24 h后葡萄糖刺激下的胰岛素分泌曲线; B图为小鼠胰岛体外培养4 d后葡萄糖刺激下的胰岛素分泌曲线。

Figure 5. Insulin functional evaluation of mouse islets after in vitro culture

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