脂肪源性干细胞对孕期LPS致子代高血压的作用及机制研究

Hypertension starts with endothelial dysfunction and progresses gradually to hypertrophy and remodelling of both the cardiac cavities and the peripheral vessels. Repairing endothelial cells and improving its function is the key therapy. Previous study in our laboratory found that prenatal exposure to lipopolysaccharide results in hypertension in adult offspring rats. The possible mechanism is that, inflammation imduced by lipopolysaccharide might have affacted the renal development of fetus and activated renal renin-angiotensin system. All these result in injury of vascular endothelium and renal epithelium. Under such condition, traditional drug therapy cannot cure hypertension fundamentally. Adipose-derived mesenthymal stem cells have Multiple differentiation potential, being able to reduce inflamotary immune response and repaire tissue by differentiation into endothelial progenitor cells. In the present study, rat hypertension was induced by prenatal exposure to lipopolysaccharide; adipose-derived stem cells were to be isolated, cultivated and identified in vitro and to be transfected with green lentivirus which express green fluorescent protein (GFP), and labeled with superparamagnetic iron oxide particles(SPIO). Transplantation of adipose-derived stem cells will be performed at the early period of pregnancy or in offspring rats when they were 3-month old. Nuclear magnetic resonance imaging technology will be used to trace the location of the transplanted stem cells.Immunofluorescence will be used to test the stem cells in kidney and mesenteric vascular positioning. Besides, blood pressure, renal function and RAS components will be tested. The aim of this study is to explore the role of transplantation of stem cells in the hypertension induced by prenatal exposure to lipopolysaccharide and the possible mechanism, providing evidences for a better therapeutic strategy of hypertension.

高血压始于内皮细胞功能紊乱及心脏和周围血管的进行性重构，修复内皮细胞、改善内皮功能是治疗的关键。我们前期研究发现，孕期脂多糖刺激引起子代大鼠高血压，孕期炎症刺激导致胚胎肾脏发育不良以及出生后RAS激活导致血管和肾小管内皮损伤可能为高血压发生的主要原因，用传统的药物对症治疗方法不能从根本上解决问题。脂肪源性干细胞具有多分化潜能，可抑制炎症免疫反应，而且可分化为内皮祖细胞修复内皮组织。本项目拟通过复制孕期脂多糖刺激致子代大鼠高血压和肾损害模型，体外分离、培养、鉴定脂肪源性干细胞，慢病毒转染绿色荧光蛋白、SPIO标记干细胞，在孕期和子鼠一月龄时移植干细胞，应用核磁共振成像技术跟踪干细胞在活体内的分布、免疫荧光法测干细胞在肾脏和肠系膜血管的定位，并通过检测血压、肾功能以及炎症因子相关指标，研究脂肪干细胞移植对孕期脂多糖刺激致子代大鼠高血压和肾损害中的作用及机制，为高血压的预防和治疗提供新的思路。

为了研究孕期脂多糖（LPS）刺激致子代大鼠高血压的发病机制以及ADSCs治疗高血压的作用机制，我们成功从SD大鼠脂肪组织中分离、培养并鉴定脂肪源性干细胞（ADSCs），并在此基础上进行体外ADSCs诱导分化为内皮祖细胞。选取64只孕鼠分为四组，分别为LPS组：于大鼠孕第8、10、12天腹腔注射0.79 mg/kg LPS，对照组：腹腔注射0.79 mg/kg生理盐水，ADSCs组：于大鼠孕第12天经尾静脉注射4.5×10^6个SPIO标记的ADSCs，LPS+ADSCs组：参见LPS组、ADSCs组。我们构建了孕期LPS刺激致子代大鼠高血压模型，于孕第12天行大鼠MRI活体示踪ADSCs在体内的定植部位，并以尾套法测量6～16周子代大鼠的血压值，分别使用HE染色、免疫组化、Western blotting和ELISA检测各项指标。动物MRI结果示，ADSCs成功定植于胚胎中。与正常组相比，LPS组8～16周子代大鼠的血压值明显升高，差异具有统计学意义（P<0.01）；与LPS组相比，LPS+ADSCs组8～16周子代大鼠的血压值明显降低，差异具有统计学意义（P<0.05）。子代大鼠第7、16周时，LPS致肾脏血管紧张素II蛋白表达升高、血管紧张素II阳性细胞数目增加，单核/巨噬细胞、淋巴细胞数目等增加；子代大鼠第1天、7周时，LPS引起肾脏凋亡细胞数目增加，而ADSCs可以缓解LPS所致的改变。各组7、16周子代大鼠的血清肾素活性、血管紧张素II以及肾功能的水平，其差异没有统计学意义（P>0.05）。以上结果说明，孕期LPS刺激致子代高血压，而ADSCs可以通过子代大鼠的肾内RAS系统缓解其高血压的发生程度。该研究为高血压的预防和治疗提供了一种新的思路。