【作者】 赵增明；

【导师】 杨晓；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2009， 博士

【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。更多还原

【Abstract】 The gastric epithelium cells are highly turnover, different cells have different refresh cycles, and the whole epithelium is in a dynamic and delicate balance, the breakout of this balance leads to various diseases happened. The gastric mucosa is composed of lots of gastric unit, each unit is composed of pit cells, parietal cells, chief cells, mucous neck cells, multipotent stem cells and minor enteroendocrine cells, all these cells are derived from the mutipotent stem cells which located at the isthmus regions. Different cells have different functions and different refresh cycles, the changes of the functions and refresh cycles of the gastric epithelium cells effect the stabilization of the gastric mucosa directly. The disorders of differentiation, proliferation and apoptosis of the epithelium cells are the reasons of kinds of gastric diseases.Pit cells are localized to the edge of the gastric mucosa, serving as the first protective barrier of the mucosa. They secrete mucus to protect the gastric epithelium from high salt, acid and pepsin. Mucus-producing pit cells are derived from the stem cells located at the isthmus, they turn over every 3 days. Parietal cells are highly differentiated, located at the isthum and the body of the gastric mucosa gland. The main function of parietal cells is secreting acid. The turnover time of parietal cells is about 54 days, they composed about 30% of the gastric epithelium cells, and they are the largest and most cells of the gland. Chief cells, also named as zymogenic cells are located at the bottom of the gland,they turn over every 190 days. Chief cells secrete pepsinogen to the lumen and activated by the acid secreted by parietal cells. These three cell lineages are the principal cells of the gastric gland.The Cre-loxP system provides a powerful means of studying genes function in specific tissues and specific cells. But, the gastric epithielium cells cpecific Cre transgenic mice is still very rare, and this hampered us to beter understand the development of the gastric, and also hampered us to study the mechanism of gastric diseases. To better understand the function of gastric epithelium cells in the homeostasis maintenance of the gastric mucosa, also to better understand their functions in the progression of gastric diseases, we established three transgenic mouse lines in which the Cre recombinase were expressed in pit cells, parietal cells and chief cells, respectively.The use of tissue specific promoter is the key of establishment of Cre transgenic mice. Calpains are Ca2+-regulated cysteine proteases. Mammals have 14 calpain genes, of which 7 are predominantly expressed in specific organs. NCL-2/calpain-8 (Capn8) has been identified as a stomach-specific calpain, whose expression is strictly localized at gastric pit cells.β-subunit of H+, K+-ATPase(Atp4b) is reported to be a parietal cells marker. Pepsinogen C (Pgc) was expressed specially in gastric chief cells, and was used as a chief cell marker. So, we used the promoters of Capn8, Atp4b and Pgc to drive the expression of Cre recombinase specially in pit cells, parietal cells and chief cells, respectively.The 3.9-kb promoter of Capn8 gene and 2.1-kb promoter of Pgc gene and 1.0-kb promoter of Atp4b gene were obtained from mouse genomic DNA by PCR amplification. The PCR products were inserted into the cloning sites of an engineered vector containing Cre coding region and hGH polyadenylation signal. The final transgenic constructs consisted of 5′sequence of promoter regions and 1.2-kb of the Cre coding region followed by the 2.1-kb of hGH polyadenylation signal. After linearized, the inserts were microinjected into the pronuclei of fertilized FVB mouse oocytes to generate the transgenic mice. Identifying the offsprings of the transgenic mice, we established three transgenic mouse lines: Capn8-Cre, Atp4b-Cre and Pgc-Cre.To check the tissue distribution and the excision activity of the Cre recombinase, the transgenic mice were crossed with a mouse strain that carries the Smad4 conditional alleles (Smad4Co/Co). The Cre-mediated excision of exon 8 of Smad4 in different tissues of the Capn8-Cre;Smad4Co/+offsprings were evaluated by PCR. Genomic DNAs were extracted from different tissues, and PCR analysis revealed Cre-mediated recombination in the stomach of all the three transgenic mice. In the Capn8-Cre;Smad4Co/+ mouse, the Cre mediated recombination also can be detected in the liver and skin tissues. To identify the exact cell types in which Cre recombinase performs its excision function, we bred the Capn8-Cre transgenic mouse with the reporter mouse ROSA26. LacZ activity was detected specially in the pit cells of the Capn8-Cre;ROSA26 double transgenic mice, In addition, Cre activity was also detected in a small subpopulation of keratinocytes located in the interfollicular epidermis of the skin and hepatocytes of the Capn8-Cre;ROSA26 transgenic mice. Furthermore, imunohistochemical staining of Smad4 in the corpus region of the Capn8-Cre;Smad4Co/Cotransgenic mouse, showed that the expression of Smad4 was significantly decreased in the pit cells.Using the same methord we identified the tissues that expressed the Cre recombinase of Pgc-Cre and Atp4b-Cre mouse. In the Pgc-Cre mouse the Cre mediated recombination can be detected in the gastric chief cells and duodenum. In the Atp4b-Cre transgenic mouse, the Cre-mediated recombination only can be detected in the gastric parietal cells.To verifyication the efficiency of the Cre recombinase of Capn8-Cre and Pgc-Cre mice, we bred these transgenic mice with a mouse strain that carries the Smad4 conditional alleles (Smad4Co/Co), and we got pit cells and chief cells specific Smad4 knockout mice: Capn8-Cre;Smad4Co/Co and Pgc-Cre;Smad4Co/Co. There are some histological differences was observed between the Smad4 knockout mouse and the control mouse.In conclusion, we established three transgenic mouse lines: Capn8-Cre, Atp4b-Cre and Pgc-Cre, using 3.9-kb promoter of Capn8 gene, 1.0-kb promoter of Atp4b gene and 2.1-kb promoter of Pgc gene, respectively. The Cre recombinase of the three transgenic mouse line can mediated efficient recombination specially in gastric pit cells, parietal cells and chief cells. The three transgenic mouse lines provide us useful tools for study the mechanism of the differentiation and genetic control of gastric epithelium cells.更多还原