γ-氨基丁酸对肠干细胞增殖分化的调节及在肠损伤中的保护作用的探究

发布时间：2018-03-23 13:00

本文选题：γ氨基丁酸　切入点：肠干细胞　出处：《山东大学》2016年硕士论文

【摘要】：研究目的：癌症研究已经在预防、早期诊断以及寻找恶性肿瘤特定的分子标记上获得了突出的成果。然而,如何防止因为使用化疗手段治疗晚期肿瘤转移所引起的正常组织受损仍然有着很大的挑战。γ氨基丁酸可以抑制神经干细胞增殖,而本研究发现肠上皮表达γ氨基丁酸能信号系统,并且丫氨基丁酸参与调节肠上皮干细胞的增殖与分化。于是,我们尝试检测是否通过药物干预γ氨基丁酸能信号系统可以促进肠干细胞增殖来加速肠上皮的由于化疗导致的损伤修复。研究方法：利用免疫组织化学技术确认γ氨基丁酸A型及B型受体亚基、合成酶、转运体和降解酶在小鼠及人肠上皮的表达分布；给予成年C57小鼠γ氨基丁酸A型受体激动剂muscimol、拮抗剂bicuculline、B型受体激动剂baclofen及拮抗剂CGP54626腹腔注射2周,取空肠组织做HE染色观察肠形态,免疫组化染色分析比较暂时性增殖细胞数目、BrdU+细胞数目、杯状细胞数目及凋亡细胞数目；给予成年C57小鼠连续5天治疗剂量或单次大剂量5氟尿嘧啶腹腔注射建立肠损伤模型,同时注射bicuculline或者CGP54626,取空肠组织做HE染色观察肠形态,免疫组化染色分析比较暂时性增殖细胞数目、BrdU+细胞数目、杯状细胞数目、凋亡细胞数目及γH2AX+细胞；利用免疫组化染色β-catenin探究Wnt信号通路,使用QPCR技术检测常备干细胞标志物lgr5、 olfm4与储备干细胞标志物]mTert、bmi mRNA相对表达量以及DNA损伤相关基因的mRNA相对表达量。研究结果：1. GAD65、GAD67、GAT3、GABAARa1、β1/2/3、GABABRI、 GABABR2在肠上皮都有表达,且在隐窝表达较多,此外GABAARπ、α5、δ γ2、GAT2均在肠上皮表达,而ABAT几乎不在肠上皮表达。2.腹腔注射muscimol减少了绒毛长度、暂时性增殖细胞、杯状细胞的数目,增加了凋亡细胞的数目；bicuculline增加了绒毛长度、杯状细胞、潘氏细胞数目;baclofen减少了暂时性增殖细胞数目而CGP54626则增加了暂时性增殖细胞数目。3.在化疗药物诱导的肠损伤模型中腹腔注射bicuculline或CGP54626相较生理盐水减少了绒毛受损,增加了暂时性增殖细胞数目,减少了γH2AX+细胞的数目,olfm4、lgr5的mRNA相对表达量较高,而bmi1、mTert的mRNA相对表达量较低,注射CGP54646同时减少了干细胞的凋亡细胞数目,且gtf2h2与prkdc的mRNA表达量下降。结论：1.γ氨基丁酸能信号系统存在于肠道上皮。2.γ氨基丁酸通过其A型受体参与调控肠上皮细胞增殖、凋亡与分化。3.γ氨基丁酸通过其B型受体参与调控肠上皮细胞增殖。4.抑制γ氨基丁酸A型受体或者B型受体可以保护化疗药物导致的肠损伤。
[Abstract]:Research objective: cancer research has achieved significant results in prevention, early diagnosis and the search for specific molecular markers for malignant tumours. However, How to prevent normal tissue damage caused by chemotherapy in the treatment of advanced tumor metastasis remains a challenge. GABA can inhibit the proliferation of neural stem cells. In this study, we found that intestinal epithelium expressed gamma-aminobutyric acid signaling system, and ABA was involved in regulating the proliferation and differentiation of intestinal epithelial stem cells. We try to detect whether drug intervention in the gamma-aminobutyric acid signaling system can promote the proliferation of intestinal stem cells to accelerate chemotherapy-induced damage repair of intestinal epithelium. Gamma aminobutyric acid type A and B receptor subunits, Expression and distribution of synthetase, transporter and degradase in mouse and human intestinal epithelium, muscimol, an agonist of gamma-aminobutyric acid type A receptor in adult C57 mice, baclofen, an antagonist, and CGP54626, an antagonist, were injected intraperitoneally for 2 weeks. Jejunum tissues were stained with HE to observe the shape of intestine. The number of temporary proliferative cells and the number of BrdU cells, goblet cells and apoptotic cells were analyzed by immunohistochemistry. Adult C57 mice were given intraperitoneal injection of 5-fluorouracil for 5 days or a single dose of 5-fluorouracil to establish a model of intestinal injury. Jejunum tissues were taken for HE staining to observe the shape of the intestine, and bicuculline or CGP54626 were injected simultaneously. The number of BrdU cells, goblet cells, apoptotic cells and 纬 H2AX cells were analyzed by immunohistochemical staining. 尾 -catenin immunohistochemical staining was used to explore the Wnt signaling pathway. QPCR technique was used to detect the relative expression of mTertnbmi mRNA and DNA damage related gene by QPCR. The results were as follows: 1. GAD65GAD67GAD67GAD67GAD67GABAARA 1, 尾 1 / 2 / 3 GABABRI, GABABR2 were expressed in intestinal epithelium, and expressed more in crypt. In addition, GABAAR 蟺, 伪 5, 未纬 2GAT2 were expressed in intestinal epithelium, while ABAT was hardly expressed in intestinal epithelium. Intraperitoneal injection of muscimol reduced villi length, temporary proliferative cells, goblet cells, increased the number of apoptotic cells and increased the length of villi and goblet cells. In the model of intestinal injury induced by chemotherapeutic drugs, bicuculline or CGP54626 were injected intraperitoneally to reduce villi damage, but CGP54626 increased the number of transient proliferating cells. It increased the number of temporary proliferative cells and decreased the number of 纬 H2AX cells. The relative expression of mRNA was higher in 纬 H2AX cells, but the relative expression of mRNA in bmi1mTert was lower. CGP54646 injection also decreased the number of apoptotic cells of stem cells. The expression of mRNA in gtf2h2 and prkdc decreased. Conclusion: 1. Gamma-aminobutyric acid signaling system exists in intestinal epithelium. 纬 -aminobutyric acid is involved in regulating the proliferation of intestinal epithelial cells through its type A receptor. Apoptosis and differentiation. Gamma-aminobutyric acid participates in regulating the proliferation of intestinal epithelial cells by its type B receptor. Inhibiting GABA type A receptor or type B receptor can protect the intestinal injury induced by chemotherapeutic drugs.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574

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