Objective To study the effects of activated signal transduction and activators of transcription 3 (STAT3) on c-jun mRNA expression and proliferation of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia in rats. Methods After PASMCs were cultured with AG490 and exposed to hypoxia, the tyrosin-phosphorylated STAT3 protein was determined in the 2 nd , 4 th , 6 th , 8 th and 12 th hour after hypoxic exposure with Western blotting. The expression of c-jun mRNA was observed with sqRT-PCR. Flow cytometric DNA analysis was used to observe the cell cycles. Results The level of tyrosin-phosphorylated STAT3 began to increase in the 6 th hour after hypoxic exposure and reached the peak of increase in the 12 th hour. The level of tyrosine-phosphorylated STAT3 and c-jun expression mRNA in the AG490-treated PASMCs was significantly lower than that in the control group. The AG490-treated PASMCs were increased in G 1/G 0 phase and decreased in the S+G 2/M phase under normoxic and hypoxic conditions. Conclusion The activated STAT3 and c-jun gene might play an important role in the early stage of hypoxia-induced proliferation of PASMCs. AG490 might inhibit c-jun expression through the lowering of the level of tyrosine phosphorylation of STAT3 which prevents the proliferation of hypoxia-induced proliferation of PASMCs.

OBJECTIVE:To explore the effects of suppressors of cytokine signaling (SOCS)3 gene on expression of c-fos, c-jun mRNA and proliferation of rat pulmonary arterial smooth muscle cells(PASMCs) under hypoxia.METHODS:PASMCs were co-transfected with pEFSOCS3 and pSV2neo by liposome, and then expression of SOCS3 protein was detected by immunocytochemistry. After PASMCs were exposed to normoxic and hypoxia at various time points respectively, expression of c-fos and c-jun mRNA was assessed by semi-quantitive RT-PCR. Flow cytometric DNA analysis was used to detect cell cycles.RESULTS:Expression of SOCS3 protein was confirmed by Western blot in PASMCs transfected with SOCS3 gene. The c-fos mRNA level in control cells peaked at 2 h of hypoxia and declined at 4 h, then peaked at 8 h secondly and declined at 12 h. C-fos mRNA level in SOCS3 gene-transfected cells at 2 h and 8 h exposed to hypoxia was lower than that in control cells at the same time points respectively (P < 0.01). The c-jun mRNA level increased at 2 h after exposure of control cells to hypoxia, peaked at 6 h of hypoxia and declined to the basal levels at 12 h. C-jun mRNA level of SOCS3 gene-transfected cells at 2 h, 4 h, 6 h, 8 h under hypoxia was lower than that in controls cells at the same time points. Compared with control PASMCs, cells in transfected with SOCS3 gene at G(1)/G(0) phase increased and those at S + G(2)/M phase decreased under normoxic and hypoxia (P < 0.01).CONCLUSION:Hypoxia induced expression of c-fos and c-jun genes, which might play an vital role in the early stage of PASMCs proliferation. SOCS3 protein may inhibit proliferation of PASMCs by lowering the tyrosine-phosphorylated level of STAT3 protein under hypoxia.

血红素氧合酶 1(HO-1)为诱导型,生理状态下,在网状内皮细胞系统和骨髓表达,在肝脏、脾脏中以高浓度存在,而又以脾脏组织活动最高,约为肝脏的10倍.可以被多种物质或刺激因素诱导表达,如热休克、缺血、辐射、低氧、高氧、重金属盐等.HO-1基因内含有HSE元件,是一种热休克蛋白,又称HSP32.目前研究证实HO系统有三种同工酶:HO-1、HO-2和HO-3.HO-2为结构型,除了化学诱生剂肾上腺皮质激素有诱导作用外,一般处于稳定状态不能被其他因素诱导,主要分布在脑组织、睾丸组织中,由CO发挥神经递质的功能,也与精子的产生和成熟有关.

目的 克隆大鼠肺动脉平滑肌细胞信号传导与转录活化因子亚型Ⅱ基因 （STAT2 ）并对此序列进行同源性分析。方法 单纯胶原酶法培养大鼠肺动脉平滑肌细胞 ,采用RT PCR克隆大鼠肺动脉平滑肌细胞信号传导与转录活化因子亚型Ⅱ基因 （STAT2 ）cDNA编码序列 ,应用PCR、四色荧光和双脱氧终止法测序 ;并对克隆序列进行同源性分析。结果 应用RT PCR克隆出大鼠肺动脉平滑肌细胞STAT2cDNA编码序列。大鼠肺动脉平滑肌细胞STAT2cDNA编码序列与Gen bank中小鼠STAT2cDNA相应序列具有高度同源性。结论 正常培养的大鼠肺动脉平滑肌细胞表达信号传导与转录活化因子亚型Ⅱ基因 （STAT2 ）。而克隆STAT2有利于探索大鼠血管性疾病信号转导的机制和防治

目的研究脂多糖 (Lipopolysaccharide, LPS) 对复合培养的微血管内皮细胞中Janus激酶(Janus kinase, JAK)基因表达的影响.方法与肺动脉平滑肌细胞复合培养鼠肺微血管内皮细胞,采用半定量RT-PCR技术检测正常组、LPS 2、8、16 h组的JAK2和TYK2基因的表达水平.结果 LPS复合培养2 h组肺微血管内皮细胞中JAK2 mRNA表达水平升高,在LPS 8 h组达最高,与正常组比较均有明显差别.在正常组和LPS刺激组均未见肺微血管内皮细胞表达TYK2基因.结论肺微血管内皮细胞不表达TYK2基因;LPS可使复合培养的肺微血管内皮细胞中JAK2基因的表达升高.

目的研究低氧对肺动脉平滑肌细胞中信号转导及转录激活因子(STATs)基因表达的影响.方法低氧处理培养的大鼠肺动脉平滑肌细胞,采用半定量RT-PCR方法检测常氧组、低氧2、6、12 h组的STAT3和STAT5基因的表达水平.结果低氧培养2 h组肺动脉平滑肌细胞中STAT3、STAT5 mRNA表达水平升高,在低氧6 h组达最高,与正常组比较均有明显差别.低氧12 h组的表达量低于6 h组,但高于正常组.结论低氧增强培养的肺动脉平滑肌细胞中STATs基因的表达,在低氧所致的肺肺动脉收缩等疾病中可能发挥重要作用.

目的探讨肺微血管内皮细胞(LMVEC)与肺动脉平滑肌细胞(PASMC)复合培养时LMVEC对PASMC周期的影响.方法 LMVEC滤膜培养后,与PASMC复合培养,采用流式细胞仪检测PASMC周期的变化.结果不同明胶浓度处理滤膜、不同种植密度相同、不同孵育时间处理滤膜对LMVEC生长数量有明显的影响,但不同孔径滤膜对LMVEC生长的影响无显著性差异.用1‰明胶处理滤膜, 105/cm2的种植密度及孵育2 d即可获得较好效果.复合培养后,PASMC的G1期细胞数减少,S+ G2/M期细胞数增多,即促进细胞生长;使LMVEC的G1期细胞数增多,S+G2/M期细胞数减少,即抑制细胞生长.结论复合培养后,促进PASMC细胞生长,抑制LMVEC细胞生长;但对两种细胞的凋亡影响不明显.

目的研究肺微血管内皮细胞(LMVEC)系培养方法并探讨脂多糖(LPS)对LMVEC酪氨酸激酶受体(TKR)活性的作用.方法贴块法培养大鼠LMVEC,并进行系列鉴定.采用放射免疫技术检测正常组、LPS(1μg/ml)作用2、8、16 h组的TKR活性的变化水平.结果获得的LMVEC具有规律的鹅卵石形态,对异植物血凝素结合试验及CD31相关抗原免疫组化染色为阳性.LPS(1 μg/ml)作用2 h组LMVEC的TKR活性升高,8 h组达最高,16 h组的表达量低于8 h组,与正常组比较均有明显差别(P<0.05).结论脂多糖(LPS)作用后LMVEC胞浆TKR活性升高,而胞膜TKR活性降低.提示在LPS所致的急性肺损伤等发病机制中TKR及其相关的信号途径可能发挥重要调节作用.

目的研究低氧对复合培养的平滑肌细胞中Janus激酶(JAK)基因表达的作用.方法鼠肺微血管内皮细胞与肺动脉平滑肌细胞复合培养,采用半定量RT-PCR技术检测常氧组、低氧2、6、12 h组的JAK1、JAK2和JAK3基因的表达水平,并对PCR产物进行测序.结果低氧复合培养2 h组肺动脉平滑肌细胞中JAK1、JAK2、JAK3 mRNA表达水平升高,在低氧6h组达最高,与正常组比较均有明显差别.低氧12 h组的表达量低于6 h组,但高于正常组.RT-PCR产物直接测序证实扩增产物片断与Genebank中相应序列相同.结论低氧增强复合培养的肺动脉平滑肌细胞中JAK基因的表达,而JAK基因在低氧所致的肺动脉收缩和低氧肺动脉高压的信号转导中可能发挥重要作用.

AIM: To study the expression level of heme oxygenase -1 and -2 in pulmonary arterial smooth muscle cell(PASMC) treated with carbon monoxide(CO). METHODS: (1) The expression of HO in cultured rat PASMC was determined by immunohistochemistry stain. (2) The levels of HO mRNA and protein were evaluated by reverse transcription- polymerase chain reaction(RT-PCR) and Western blot. RESULTS: (1) PASMC cytoplasm showed yellow (++-+++) in hypoxia group, the buffy color(++++) in CO group as cells were stained by HO-1 antibody. (2) The expression level of HO-1 mRNA (1.25±0.37) in hypoxia group was higher than that of normal group (0.12±0.04). It was much higher (3.52±0.68) in CO group, than that of hypoxia group. (3)The content of HO-1 protein (1.07±0.15) in hypoxia 48 h group was higher than that of hypoxia 24 h group(0.52±0.04). It was higher(3.65±0.43) in CO group than that of hypoxia group.It was highest in CO 48 h group. CONCLUSION: Low density of CO and hypoxia enhanced the expression of HO-1 mRNA and protein in PASMC when cell were treated in hypoxia or CO,indicating that HO-1 has regulatory effect on PASMC during hypoxia.

细胞因子作用的靶细胞中存在的蛋白酪氨酸激酶(PTK)可以介导细胞因子与其受体结合后的信号蛋白分子级联活化反应.JAK(Janus kinase)是一种重要的PTK.细胞因子与受体结合后激活JAK,进而激活“信号转导子和转录激活子”(signal transducer and activator of transcription,STAT),应激反应也能激活JAK-STAT信号传导,再诱导目的基因表达.近来研究表明,JAK-STAT途径是人体内生理和病理反应的共同通路之一,与多种疾病发病及防治密切相关.

目的通过研究CO和低氧作用后大鼠肺血管细胞增殖和凋亡状况,探讨低氧肺动脉高压的发病机制及防治措施.方法应用免疫组织化学、原位末端标记及Western杂交等方法检测常压低氧大鼠肺血管壁细胞增殖、凋亡及c-myc基因的表达状况.结果正常组、低氧和锡原卟啉组、低浓度CO和血晶素组大鼠肺动脉均存在增殖和凋亡的阳性细胞,两类细胞在肺内呈不均匀散在分布.低氧和锡原卟啉组大鼠肺血管增殖细胞数显著升高而凋亡细胞数显著减少,细胞增殖凋亡比值分别为对照组的5倍或4倍,而低浓度CO和血晶素组肺血管增殖细胞和凋亡细胞系数均显著增加,细胞增殖凋亡比值均为1.2.c-myc在低氧和锡原卟啉组大鼠肺内表达显著增加,在低浓度CO和血晶素组大鼠肺内表达减少.结论增殖和凋亡现象共存在于正常和处理大鼠的肺血管细胞中,也许c-myc等基因的异常表达导致了细胞增殖和凋亡的失衡,进而调节了慢性低氧肺血管结构的改建.

Flow cytometry and immunohistochemistry was used to investigate of the affects on expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) in rat pulmonary microvascular endothelial cell (PMVEC) by TNF-α and IL-1. It was showed that TNF-induced PECAM-1 proein expression reduction, but IL-1 did not affect significently in PMVEC. These results suggest that inflammation-stimuli can cause reduction and redistribution of PECAM-1 expression on PMVEC, and PECAM-1 may play an impotant action during inflammation.