胃粘膜上皮细胞特异性表达Cre重组酶转基因小鼠的构建

发布时间：2018-05-14 03:39

本文选题：顶细胞 + 壁细胞　；参考：《中国人民解放军军事医学科学院》2009年博士论文

【摘要】： 胃粘膜上皮细胞具有很高的更新速度,各种上皮细胞具有不同的代谢周期,处于一种动态的、精致的平衡之中,这种平衡的打破导致各种疾病的发生。胃粘膜由许多胃单位构成的,每个胃单位由顶细胞(pit cells)、壁细胞(parietal cells)、主细胞(chief cells)、颈粘液细胞(mucous neck cells)、多能干细胞(multipotent stem cells)以及少量的内分泌细胞(enteroendocrine cells)等多种上皮细胞组成,其中顶细胞、壁细胞、主细胞是胃粘膜最主要的三种细胞。各种上皮细胞均来源于峡部的多能干细胞,每种细胞具有不同的功能和更新速率。细胞分化、增殖和凋亡的改变以及更新速度的改变直接影响到胃粘膜结构的稳定,是导致胃相关疾病发生的原因。顶细胞位于粘膜层的最外侧,与胃腔的外界环境直接接触,表面覆盖着从细胞释放出来的粘液,有保护细胞免受胃液内高浓度盐酸和胃蛋白酶损伤的作用,是胃粘膜的第一道屏障,每3天更新一次。壁细胞属于高度特化的终末细胞,主要功能是分泌盐酸,位于胃腺腔的颈部和体部,约占胃粘膜上皮细胞总数的30%,每隔54天更新一次,是上皮细胞中体积最大、数量最多的细胞。主细胞又称胃酶细胞(zymogenic cells)位于胃腺腔的底部,约190天更换一次,可分泌胃蛋白酶原,并被壁细胞分泌的盐酸激活,成为具有活性的胃蛋白酶。这三种细胞占粘膜层上皮细胞的绝大部分,是粘膜层的主要细胞成分。基于Cre-LoxP系统的组织特异性条件基因敲除技术是研究基因在特定组织、特定细胞生理功能的有效手段。目前在胃的各种上皮细胞中特异性表达Cre重组酶的转基因小鼠非常少见,在某种程度上妨碍了对调节胃正常发育以及胃相关疾病遗传机制的研究。为了深入研究调控胃粘膜层上皮细胞功能的遗传机制及其在胃粘膜稳态维持以及胃组织相关疾病中的功能,我们分别构建了在顶细胞、壁细胞、主细胞特异性表达Cre重组酶的转基因小鼠。组织细胞特异性启动子的选择是构建组织细胞特异性表达Cre重组酶转基因小鼠的关键。钙离子激活蛋白酶(Calpains)是受Ca2+调节的半胱氨酸蛋白酶家族,该家族在哺乳动物中有14个成员,其中的7个表达具有明显的组织特异性。NCL-2/calpain-8 (Capn8)在胃粘膜层顶细胞特异性表达。H+,K+-ATP酶β亚单位(β-subunit of H+, K+-ATPase,Atp4b)是壁细胞的标志物分子。胃蛋白酶原C(Pepsinogen C,Pgc)在胃粘膜的主细胞特异性表达,是主细胞的标志物分子。因此,本研究拟分别采用Capn8,Atp4b和Pgc基因的启动子,实现Cre重组酶在顶细胞、壁细胞和主细胞的特异性表达。我们利用PCR扩增的方法获得了3.9-kb Capn8和1.0-kb Atp4b以及2.1-kb Pgc基因的启动子。将获得的启动子连入含有1.2-kb编码Cre重组酶的基因和2.1-kb人生长激素基因多聚腺苷酸加A信号(hGH)的载体中,得到转基因载体。转基因载体经限制性内切酶线性化后,经显微注射引入小鼠受精卵。将显微注射后的受精卵移植入假孕母鼠,从子代小鼠中鉴定得到Capn8-Cre,Atp4b-Cre和Pgc-Cre三种转基因小鼠。通过将三种胃组织特异性Cre转基因小鼠与Smad4条件基因打靶小鼠(Smad4Co/Co)和ROSA26报告小鼠杂交检测Cre重组酶表达的时空分布。提取Capn8-Cre;Smad4Co/+小鼠各组织基因组DNA,用Smad4基因特异引物进行PCR,检测Cre重组酶介导的重组及其组织特异性。结果显示,Cre重组酶可在胃介导Smad4基因的敲除并产生特异的阳性条带。此外,在肝脏和皮肤也有Cre重组酶的表达。Capn8-Cre;Smad4Co/Co小鼠各组织基因组DNA的Southern Blot杂交鉴定结果也证实Cre重组酶在胃、肝脏及皮肤中表达。通过LacZ染色检测Capn8-Cre;ROSA26双转基因小鼠中Cre重组酶表达的组织及细胞类型。结果显示,Capn8-Cre重组酶在胃粘膜层顶细胞特异性表达。除此之外,在肝脏的肝细胞及皮肤少数角质细胞也有表达。Capn8-Cre;Smad4Co/Co基因敲除小鼠胃粘膜层Smad4免疫组织化学染色结果也显示,Capn8-Cre重组酶在顶细胞介导了Smad4的基因敲除。利用同样的方法我们检测了Atp4b-Cre和Pgc-Cre转基因小鼠Cre重组酶的组织表达谱。Pgc-Cre转基因小鼠除在胃粘膜主细胞特异性表达Cre重组酶外,在十二指肠也有表达。Atp4b-Cre转基因小鼠仅在胃粘膜壁细胞特异性表达Cre重组酶。为进一步验证Cre重组酶的有效性,我们利用Capn8-Cre和Pgc-Cre转基因小鼠与Smad4Co/Co小鼠交配,获得了胃顶细胞特异性Smad4基因敲除小鼠( Capn8-Cre;Smad4Co/Co )和胃主细胞特异性Smad4基因敲除小鼠(Pgc-Cre;Smad4Co/Co),并初步观察到Smad4基因敲除导致胃粘膜层发生的组织学改变。总之,我们利用3.9-kb Capn8基因启动子,1.0-kb Atp4b基因启动子和2.1-kb Pgc基因启动子成功地构建了在胃粘膜层顶细胞、壁细胞和主细胞特异性表达Cre重组酶的转基因小鼠。研究结果证明这三种胃组织细胞特异性Cre重组酶转基因小鼠能够介导被LoxP序列锚定的基因在相应胃粘膜细胞中被有效剔除。这些转基因小鼠的成功研制为研究胃粘膜上皮细胞的谱系分化以及相关细胞的遗传控制机制提供了理想的工具。
[Abstract]:The epithelial cells of the gastric mucosa have a high rate of renewal, and the various epithelial cells have different metabolic cycles and are in a dynamic, delicate balance. This balance breaks the occurrence of various diseases. The gastric mucosa is composed of many gastric units, each of which is pit cells, parietal cells, and main cell. (chief cells), cervical mucous cells (mucous neck cells), pluripotent stem cells (multipotent stem cells) and a small number of endocrine cells (enteroendocrine cells), such as a variety of epithelial cells, of which the top cells, wall cells, main cells are the most important three kinds of cells in the gastric mucosa. The cells have different functions and rates of renewal. The changes in cell differentiation, proliferation and apoptosis and the change of the rate of renewal directly affect the stability of the structure of the gastric mucosa, which is the cause of the occurrence of gastric related diseases.
The parietal cells are located at the most outside of the mucous layer and directly contact with the external environment of the gastric cavity. The surface covers the mucus released from the cells. It protects the cells from the high concentration of hydrochloric acid and pepsin in the gastric juice. It is the first barrier of the gastric mucosa and is updated every 3 days. Parietal cells belong to highly specialized terminal cells. The main work is the main work. The ability to secrete hydrochloric acid, located in the neck and body of the gastric gland cavity, about 30% of the total number of epithelial cells in the gastric mucosa, once every 54 days, is the largest and most large cell in the epithelial cells. The main cell is called zymogenic cells at the bottom of the gastric gland cavity and is replaced about 190 days, which can secrete pepsinogen and be thin wall. The secretion of hydrochloric acid activates and activates pepsin. These three cells account for the vast majority of mucosal epithelial cells and are the major cell components of the mucosa.
Tissue specific conditional gene knockout based on Cre-LoxP system is an effective means to study genes in specific tissues and specific cell physiological functions. At present, transgenic mice that specifically express Cre recombinant enzymes in various epithelial cells of the stomach are very rare. To a certain extent, it hinders the regulation of normal development of the stomach and the related diseases of the stomach. In order to study the genetic mechanism of the function of the epithelial cell of the gastric mucosa and its function in the homeostasis of gastric mucosa, and the function of the gastric tissue related diseases, we constructed the transgenic mice with the expression of Cre recombinant enzyme in the apical cell, the wall cell and the main cell specific expression.
The selection of tissue cell specific promoter is the key to the construction of tissue cell specific expression of Cre recombinant transgenic mice. Calcium activated proteinase (Calpains) is a family of cysteine protease regulated by Ca2+. The family has 14 members in mammals, and 7 of them have obvious tissue specific.NCL-2/calpain-. 8 (Capn8) is a specific expression of.H+ in the parietal cells of the gastric mucosa, and the K+-ATP enzyme beta subunit (beta -subunit of H+, K+-ATPase, Atp4b) is a marker molecule of the wall cells. The pepsinogen C (Pepsinogen C, Pgc) is expressed specifically in the main cells of the gastric mucosa and is a marker molecule of the main cells. The promoter of Cre can express the specific expression of the recombinant enzyme in the parietal cells, parietal cells and main cells.
We obtained the promoter of 3.9-kb Capn8 and 1.0-kb Atp4b and 2.1-kb Pgc gene by PCR amplification. The promoter connected into the vector containing 1.2-kb encoded Cre recombinant enzyme and 2.1-kb human growth hormone gene polyadenyl adenylate plus A signal (hGH). The transgenic vector was restricted by restriction endonuclease. After linearization, the mouse fertilized eggs were injected into the mice by microinjection. The fertilized eggs after microinjection were transplanted into the pregnant mice, and three transgenic mice of Capn8-Cre, Atp4b-Cre and Pgc-Cre were identified from the offspring mice.
The spatio-temporal distribution of the expression of Cre recombinant enzyme was detected by hybridization between three gastric tissue specific Cre transgenic mice and Smad4 conditional gene targeting mice (Smad4Co/Co) and ROSA26. Capn8-Cre, genomic DNA of every tissue of Smad4Co/+ mice and PCR with Smad4 specific primers were used to detect the recombination and tissue specific of Cre recombinant enzyme. The results showed that the Cre recombinant enzyme could be knocked out of the stomach mediated Smad4 gene and produce a specific positive band. In addition, the expression of the Cre recombinant enzyme was.Capn8-Cre in the liver and the skin; the Southern Blot hybridization identification results of the genomic DNA in the Smad4Co/Co mice also confirmed that the Cre heavy enzyme was expressed in the stomach, the liver and the skin. The tissue and cell types of Cre recombinant enzyme in Capn8-Cre and ROSA26 double transgenic mice were detected. The results showed that the Capn8-Cre recombinase was expressed specifically in the gastric mucosal layer apical cells. In addition, the.Capn8-Cre was expressed in the liver cells and the few keratinocytes in the skin, and the Smad4 immune group in the gastric mucosa layer of the Smad4Co/Co gene knockout mice. The results of chemical staining also showed that Capn8-Cre recombinase mediated Smad4 knockout in the apical cells.
Using the same method, we detected the tissue expression profiles of Cre recombinant enzyme of Atp4b-Cre and Pgc-Cre transgenic mice..Pgc-Cre transgenic mice expressed.Atp4b-Cre transgenic mice in the duodenum, except for the specific expression of Cre recombinant enzyme in the main gastric mucosa, and the expression of Cre recombinant enzyme in the gastric mucosa only in the duodenum.
In order to further verify the effectiveness of the Cre recombinant enzyme, we use Capn8-Cre and Pgc-Cre transgenic mice to mate with Smad4Co/Co mice, and obtain the specific Smad4 gene knockout mice (Capn8-Cre; Smad4Co/Co) and the specific Smad4 gene knockout mice (Pgc-Cre; Smad4Co/Co) of the gastric main cell (Pgc-Cre; Smad4Co/Co), and preliminarily observe the Smad4 gene knockout guide. Histologic changes in the mucosa of the gastric mucosa.
In conclusion, we successfully constructed transgenic mice with the 3.9-kb Capn8 gene promoter, the 1.0-kb Atp4b promoter and the 2.1-kb Pgc gene promoter in the gastric mucosa, the wall cells and the main cells specifically expressed the Cre recombinant enzyme. The results showed that the three gastric tissue cell specific Cre recombinase transgenic mice could mediate. The genes that are anchored by the LoxP sequence are effectively eliminated in the corresponding gastric mucosa cells. The successful development of these transgenic mice provides an ideal tool for the study of the genealogical differentiation of gastric epithelial cells and the genetic control mechanism of the related cells.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346

【引证文献】