降钙素基因相关肽对大鼠胃平滑肌的调控作用

发布时间：2018-11-06 08:54

【摘要】：目的:观察降钙素基因相关肽在体外培养细胞中的作用,探究降钙素基因相关肽对大鼠胃平滑肌的调控作用和机制。方法:幼年SD大鼠胃,采用组织碎块贴瓶底和消化酶消化两种方法培养SD大鼠胃平滑肌细胞,成功提取胃平滑肌细胞(smooth muscle cell,SMC)并用高糖DMEM营养液(含20%血清)培养。分离成功的平滑肌细胞利用可发生特异反应的α-actin细胞组化进行鉴别。实验分为:CGRPI、CGRPII、CGRPIII、CGRP8-37+CGRP组、空白对照共5组,加入相对应的试剂。利用电脑数据分析系统测量平滑肌细胞加药前后长短;用钙荧光标志剂测测胞内的Ca2+浓度;Celltiter-Glo(?)组合试剂测定细胞内ATP含量。结果:贴块法和酶解法都能提取细胞,酶解法所提细胞所用时间短,但杂质较多,贴块法虽然时间相对较长但细胞纯度相对较高。成功分离SD大鼠胃平滑肌细胞,细胞呈现梭子状或是无规则图形,细胞核处在细胞中间。免疫组化鉴定能够看到平滑肌细胞成无规则形,细胞浆呈很浅的棕黄色,细胞核呈淡蓝色,表明提取的正是实验所需的胃平滑肌细胞。CGRP在各个剂量时都能够降低胃肠平滑肌胞浆里Ca2+含量,并在一定范围内随CGRP剂量增加降低Ca2+含量作用增大。加入CGRP后和对照组相比能降低平滑肌细胞所携带的ATP,并且随着CGRP浓度的增加ATP的含量改变也增大(P0.05)。结论:应用组织碎块贴瓶底法得到的胃平滑肌细胞,通过继续培养、时差贴瓶底等可以得到所需的鼠胃SMC。由实验可以看出CGRP能松弛胃平滑肌,降低细胞内钙离子浓度及ATP含量,提示钙离子浓度变化可能参与了其松弛平滑肌细胞的机制;CGRP可能通过改变胃平滑肌细胞动能来抑制胃SM运动。
[Abstract]:Aim: to investigate the effect of calcitonin gene-related peptide (CGRP) on rat gastric smooth muscle in vitro. Methods: gastric smooth muscle cells (GSMCs) of SD rats were cultured by tissue fragmentation and digestive enzyme digestion. Gastric smooth muscle cells (smooth muscle cell,SMC) were extracted successfully and cultured with high glucose DMEM nutrient solution (containing 20% serum). The isolated smooth muscle cells were identified by histochemistry of 伪-actin cells that could react specifically. The experiment was divided into CGRPI,CGRPII,CGRPIII,CGRP8-37 CGRP group and blank control group. Computer data analysis system was used to measure the length of smooth muscle cells before and after drug addition; calcium fluorescent marker was used to measure intracellular Ca2 concentration; Celltiter-Glo (?) The content of ATP in cells was determined by combined reagent. Results: the cells could be extracted by the method of sticking and enzymatic hydrolysis. The time of the cells extracted by enzymatic hydrolysis was short, but the impurity was more. Although the time of the method was relatively long, the purity of the cells was relatively high. Gastric smooth muscle cells of SD rats were isolated successfully. The cells showed spindle shape or irregular pattern and the nucleus was in the middle of the cells. Immunohistochemical staining showed that smooth muscle cells were irregular in shape, the cytoplasm was very light brown, and the nucleus was light blue. The results showed that the gastric smooth muscle cells (GSMCs) were extracted. CGRP could decrease the content of Ca2 in the cytoplasm of gastrointestinal smooth muscle at every dose and increase the content of Ca2 with the increase of CGRP dose in a certain range. Compared with the control group, the addition of CGRP decreased the ATP, carried by smooth muscle cells and increased the content of ATP with the increase of CGRP concentration (P0.05). Conclusion: the gastric smooth muscle cells obtained by using tissue fragments and flask bottom method can be used to obtain the required gastric SMC. through continuous culture and jet-lag sticking to the bottom of the bottle. It can be seen from the experiment that CGRP can relax gastric smooth muscle and decrease intracellular calcium ion concentration and ATP content, suggesting that the change of calcium ion concentration may be involved in the mechanism of relaxation smooth muscle cells. CGRP may inhibit gastric SM motility by changing the kinetic energy of gastric smooth muscle cells.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R363

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