乙酰肝素酶对滋养细胞生物学行为的影响及其作用机制的研究

In previous study, we detected the protein expression of heparanase in placenta between severe preeclampsia and healthy controls by western blot. The result showed the expression in severe preeclampsia was obvious higher than the healthy control. In order to further observe if heparanase can effect on the biological behavior, the heparanase recombination protein was added into the culture medium 0f human trophoblastic cell line,HTR8/SVneo when performing the invasion experiment. The result showed that the invasion ability of trophoblastic cell decreased. In sum, we thought the heparanase was associated with the biological behavior of trophoblastic cell. In order to further study, we planned to construct the overexpression vector and shRNA interference vector. We would transfect the human trophoblastic cell line HTR8/Vneo,JAR and JEG-3.Then the proliferation, apoptosis, migration, invasion and tube formation of these three cell lines would be observed by method of CCK-8 kit, co-culture with hUtMVEC and RL95-2,tunel kit and so on..The biological behavior of trophoblastic cell was controlled by many cell signaling pathways, such as FAK, PI3K and JAK signaling pathway. So we hypothesized heparanase effected on trophoblastic cell by these signaling pathways. But we didn’t know which pathway played a leading role and which protein participated. In order to explore the potential mechanism of heparanase, the total RNA and protein of the transfected cell were extracted, then the expression of A-Raf、B-Raf、C-Raf、Mos、Tpl2、MLK3、TAK、DLK、MEKK1/2/3/4、ASK1、MEK1/2/5、MKK3/4/6/7、Erk1/2/5、p38 MAPKα/β/γ/δ、SAPK、JNK1/2/3 and BMK1were detected. In vivo, the vectors were injected into the placenta of pregnancy rat. Outcome of pregnancy, blood pressure and urine were detected. After 72h, newborn rat was weighed. Placenta was collected. Placenta was sliced, RNA and protein were extracted. The expression of A-Raf、B-Raf、C-Raf、Mos、Tpl2、MLK3、TAK、DLK、MEKK1/2/3/4、ASK1、MEK1/2/5、MKK3/4/6/7、Erk1/2/5、p38 MAPKα/β/γ/δ、SAPK、JNK1/2/3 and BMK1 were detected. This study would provide new data and position for mechanism of heparanase, preeclampsia, and also a new target spot.

课题组前期研究发现重度子痫前期胎盘中乙酰肝素酶的表达明显高于正常妊娠组，外源性乙酰肝素酶可降低人滋养细胞系HTR8/SVneo侵袭力，提示乙酰肝素酶与滋养细胞的生物学行为密切相关。本研究拟在前期研究的基础上，通过构建过表达和沉默表达乙酰肝素酶的稳定转染人滋养细胞系及构建孕鼠胎盘定点注射模型，采用细胞共培养、侵袭实验和体外管腔形成实验等，观察乙酰肝素酶对滋养细胞生物学行为的影响；通过荧光定量PCR和免疫印迹法等检测转染细胞系及孕鼠胎盘组织丝裂原活化蛋白激酶信号传导通路多个相关分子及局部粘附激酶等6条信号传导通路关键分子的mRNA和蛋白表达水平，明确乙酰肝素酶与丝裂原活化蛋白激酶等信号传导通路的相关性，初步探索其作用机制。本研究将为乙酰肝素酶的作用机制研究提供新的实验数据，为子痫前期等疾病的发病机制研究提供新的研究方向、研究数据和研究基础，同时也可为这类疾病提供新的治疗靶点及研究基础。

课题组前期研究发现重度子痫前期胎盘中乙酰肝素酶的表达明显高于正常妊娠组，外源性乙酰肝素酶可降低人滋养细胞系HTR8/SVneo侵袭力，提示乙酰肝素酶与滋养细胞的生物学行为密切相关。本研究拟在前期研究的基础上，通过构建过表达和沉默表达乙酰肝素酶的稳定转染人滋养细胞系及构建孕鼠胎盘定点注射模型，采用细胞共培养、侵袭实验和体外管腔形成实验等，观察乙酰肝素酶对滋养细胞生物学行为的影响；通过荧光定量PCR和免疫印迹法等检测转染细胞系丝裂原活化蛋白激酶信号传导通路多个相关分子及局部粘附激酶等6条信号传导通路关键分子的mRNA和蛋白表达水平，明确乙酰肝素酶与丝裂原活化蛋白激酶等信号传导通路的相关性，初步探索其作用机制。研究结果显示：与过表达对照相比，过表达HPSE可显著增加滋养细胞株增殖、侵袭和抗凋亡的能力，但是对滋养细胞株的成管能力无显著的干扰；与干扰对照相比，干扰HPSE的mRNA表达可显著的降低滋养细胞增殖、侵袭、成管及抗凋亡的能力。与过表达对照相比，过表达HPSE的滋养细胞内细胞因子表达降低，过表达与过表达对照相比，细胞因子蛋白表达水平比值<0.6667的细胞因子共计15种。经过生物学信息分析后发现，差异细胞因子主要涉及炎症反应、细胞凋亡、血管生成和细胞外基质的生成和分解等过程；大部分差异细胞因子均可与MMP9产生直接或间接关系，说明过表达HPSE可能通过调控MMP9的表达，影响滋养细胞的生物学功能；与干扰对照相比，HPSE干扰细胞株中磷酸化p38/总p38的比值升高，且抑制p38磷酸化后，HPSE干扰滋养细胞株的侵袭能力显著提高，说明干扰HPSE后，细胞可通过p38MAPK途径影响滋养细胞的侵袭力；与对照相比，过表达HPSE和干扰HPSE后，Rho蛋白家族、磷酸肌醇3 激酶、丝裂原活化蛋白激酶、JAK/STAT 信号传导通路、Smad 蛋白家族和Notch 信号传导通路并无显著改变。本项目研究结果提示胎盘HPSE的表达异常确实与滋养细胞生物学行为密切相关，HPSE表达的降低与子痫前期，特别是重度子痫前期的发生发展密切相关，为明确子痫前期的发病机制提供了重要的研究基础和研究方向，同时HPSE也有望成为子痫前期治疗的潜在靶点，具有较好的科学意义和一定的应用前景。