Hedgehog信号通路在海水吸入大鼠急性肺损伤中的作用及其机制研究
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摘要
背景和目的:
海水淹溺不仅是航海事故、海上生产作业、旅游的死亡原因之一,而且是军队海上训练、作战、抢滩登陆战斗和非战斗减员的重要原因。海水淹溺后绝大部分病人都有肺损伤并继发肺水肿甚至海水型急性肺损伤(SW-ALI)/急性呼吸窘迫综合征(SW-RDS),使病情更加危重,救治极为困难。海水淹溺后高度肺水肿的形成与肺泡毛细血管膜的损伤密切相关,作为肺泡毛细血管膜的内屏障,肺微血管内皮细胞(pulmonary microvascular endothelial cell,PMVEC)的增殖与修复将有利于降低肺泡毛细血管膜的通透性,从而减轻肺水肿程度。因此研究肺微血管内皮细胞损伤后修复的信号转导机制,为防治海水淹溺ALI提供了新的思路。
Hedgehog(Hh)信号通路是与胚胎形成及形态发生有关的信号途径。主要由胞外的Hedgehog蛋白、细胞膜表面的特异性受体Patched (Ptc)及相关蛋白Smoothened (Smo)、胞内的转录因子Ci/Gli组成。Hedgehog蛋白是一种分泌型蛋白,在哺乳动物中包括Shh(Sonic hedgehog)、Ihh(Indian hedgehog)和Dhh(Desert hedgehog)三种家族成员。近年发现,Hh家族蛋白在许多成熟组织也有表达,调节细胞的增殖和自我更新,参与组织的损伤修复,并与多种疾病的发生发展相关。研究表明,Hedgehog (Hh)信号通路的激活可促进微血管内皮细胞增殖,诱导微血管重建。在此基础上,我们推测海水吸入ALI时,通过激活Hh信号通路,可促进肺微血管内皮细胞增殖,有利于肺泡毛细血管膜损伤后修复,从而减轻肺水肿程度。为此,本实验拟从细胞细胞水平检测Hh信号通路激活后对肺微血管内皮细胞增殖和凋亡的影响,从动物水平检测Hh信号通路激活后对肺微血管通透性和肺水肿程度的影响,为海水淹溺ALI的防治提供新的实验依据。
方法:
1.采用外周肺组织贴块法培养大鼠PMVEC,抽取组织块进行切片观察,以大鼠肺动脉平滑肌细胞和人脐静脉内皮细胞为对照,对培养细胞进行CD34、植物凝集素BSI、Ⅷ因子相关抗原免疫细胞化学染色鉴定,通过光镜和透射电镜观察细胞形态和超微结构。
2.将大鼠PMVEC随机分为正常对照组和海水处理2h、4h、8h和16h组。RT-PCR法分别检测细胞中Shh、Ptc-1和Gli1 mRNA表达情况;Western blotting法分别检测细胞中Shh、Ptch1和Gli1蛋白表达情况;光镜和透射电镜观察细胞形态和超微结构变化。
3.将大鼠PMVEC随机分为正常对照组和Shh50 ng/ml组、Shh100 ng/ml组、Shh200 ng/ml组、Shh400 ng/ml组和cyclopamine+Shh100 ng/ml组。MTT法检测各组细胞增殖活性。
4.大鼠PMVEC随机分为正常对照组、海水组、Shh50 ng/ml +海水组、Shh100 ng/ml +海水组、Shh200 ng/ml +海水组、Shh400 ng/ml +海水组和cyclopamine+Shh100 ng/ml+海水组。MTT法检测各组细胞增殖活性,TUNNL法检测细胞凋亡情况。5.采用屏气后经气管吸入4ml/Kg海水复制大鼠海水吸入ALI模型,随机分为对照组和海水组,每组又分为1h、2h、4h和8h四个时相点检测动脉血氧分压(PaO2)和肺组织湿干重比(W/D),肺微血管通透性、肺组织MPO活性和MDA含量、血清和BALF中TNF-α和IL-1β含量、RT-PCR法和Western Blot法分别检测肺组织Shh、Ptc-1和Gli1mRNA和蛋白表达水平,并进行肺组织病理形态学观察。
6.采用海水吸入ALI大鼠模型,静脉注射人重组Shh因子6mg/kg干预后,检测大鼠PaO2、肺W/D、肺微血管通透性、Ptc-1和Gli1mRNA和蛋白表达变化,并进行病理形态学观察。
结果:
1.组织块法培养大鼠PMVEC的综合鉴定结果显示:组织块源于外周肺组织,Ⅷ因子相关抗原和CD34免疫荧光化学染色阳性,植物凝集素BSI结合试验阳性,透射电镜细胞表面有较多突起,胞浆中有较多吞饮小泡。
2.与正常对照组比较,从4h开始,海水组细胞Shh、Ptc-1和Gli1 mRNA和蛋白表达量减少,8h最低,16h有所增加。光镜下可见海水处理16h后,PMVEC细胞皱缩,胞质减少,细胞间距增大,部分细胞脱落。电镜下可见海水处理16h大鼠PMVEC细胞表面突起减少,胞浆浓缩,线粒体水肿,嵴消失,胞浆中吞饮小泡增多,核内异染色质增多。
3. Shh处理正常大鼠PMVEC细胞增殖活性较对照组明显增强,并呈剂量依赖性。Cyclopamine+Shh100ng/ml组细胞增殖活性明显低于对应时相点Shh100ng/ml组。
4.与正常对照组相比,海水组PMVEC细胞增殖明显减少,而Shh处理各组细胞增殖活性明显高于海水组;Cyclopamine组细胞增殖活性明显低于Shh处理组。海水处理PMVEC 8h后,细胞凋亡较对照组明显增加,而Shh处理各组细胞凋亡明显低于海水组,Cyclopamine组细胞凋亡明显高于Shh处理组。
5.大鼠经气管吸入海水后,其PaO2明显低于对应时相点对照组,氧合指数<300;肺W/D、肺微血管通透性、肺组织MPO活性和MDA含量、血清和BALF中TNF-α和IL-1β含量明显高于对应时相点对照组,并随时间延长逐渐增加;肺组织Shh、Ptc-1和Gli1mRNA和蛋白表达水平显著低于对照组,并随时间延长逐渐降低。
6.海水吸入ALI大鼠注射人重组Shh因子后,其PaO2明显高于海水组,肺W/D和肺微血管通透性明显低于海水组,Ptc-1和Gli1mRNA和蛋白表达明显高于海水组。
结论:
1.海水刺激下大鼠PMVEC增殖受抑制,细胞凋亡增多。
2.人重组Shh可通过激活Hedgehog信号通路,促进正常大鼠PMVEC增殖并抑制其凋亡。
3.Hedgehog信号分子在海水处理大鼠PMVEC中表达降低,采用人重组Shh激活Hedgehog信号通路可促进海水处理大鼠PMVEC细胞增殖,抑制细胞凋亡。
4.成功建立海水吸入ALI大鼠模型。海水吸入ALI大鼠肺组织中肺微血管通透性和肺水肿程度随时间延长进行性增加;Hedgehog信号分子在海水吸入ALI大鼠肺组织中表达降低,采用人重组Shh激活Hedgehog信号通路后,可降低海水吸入ALI大鼠肺微血管通透性,减轻肺水肿程度。这种效应可能是Hedgehog信号通路激活后通过对PMVEC的保护作用实现的。
Backgroud and Objective:
sea water drowning is not only an important cause of death in the accident of civil sailing, wrecking on the sea, but also the key factor of army non-military depletion of numbers in training or fighting on the sea. As the consequence or result of sea water drowning, lung injury, secondary pulmonary edema or sea water respiratory distress syndrome(SW-RDS) occurs in most of patients, and leads to the challenge of treatment. Alveolus capillary barrier injure plays a key role in the process of severe pulmonary edema. The proliferation and repair of Pulmonary microvascular endothelial cells can improve the permeability of the barrier and alleviate the degree of pulmonary edema. Therefore, the understanding of signaling mechanism involved in PMVEC reparation after sea water drowning injure will provide new treatment strategies.
The Hedgehog signaling pathway plays a fundamental role in the development of various embryonic tissues development. It is composed of Hh protein、the transmembrane receptor protein patched (ptc) and smoothend(smo) and the transcription factor Gli. Hh is a protein secreted by cells. There are three kinds of it: Sonic Hedgehog(Shh)、Indian Hedgehog (Ihh) and Desert Hedgehog(Dhh).Recent investigations found that main component molecules of Hh pathway are present in many mature tissue and regulat cell proliferation and renewal in the process of repairation after injure. The activation of Hh signaling pathway can increase PMVEC proliferation and induce capillary remodeling. Thus we hypothesize that activation of Hh pathway can improve the PMVEC proliferation (survival) and reparation in the process of sea water drowning induced lung injure. Therefore we investigate the effect of Hh signaling pathway on proliferation and apoptosis of PMVEC, and the improvements of pulmonary microvascular permeability and pulmonary edema in rat sea water drowning model.
Methods:
1. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Histological sections from peripheral lung tissue pieces used for cell culture were examined. Used rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as control, CD34, lectin from Bandeiraea simplicifolia and factorⅧrelated antigen in the cultured cells were quantified by immunocytochemical staining. In addition, the cell morphology and ultrastructure were observed with inverted optical microscope and transmission electron microscope respectively.
2. PMVEC were divided randomly five groups:normal control, sea water treatment 2h、sea water treatment 4h、sea water treatment 8h and sea water treatment 16h. The expression of Shh and Ptc-1 mRNA was detected by RT-PCR and the protein expression by Western blot . The changes of cell morpha and ultrastructure were investigated with microscope or electron microscope.
3. PMVEC were divided randomly sis groups: normal control, Shh 50 ng/ml treatement group, Shh 100 ng/ml treatement group, Shh 400 ng/ml treatement group,and cyclopamine treatment group.MTT was used to detect the proliferational activity of PMVECs.
4. PMVEC were divided randomly seven groups: normal control, sea water treatment group、Shh 50 ng/ml+sea water treatment group、Shh 100 ng/ml+sea water treatment group、Shh 200 ng/ml+sea water treatment group、Shh 400 ng/ml+sea water treatment group,and cyclopamine +sea water treatment group. MTT was used to detect the proliferational activity of PMVEC.TUNNL was used to examinate the apoptosis of PMVECs.
5. A rat model of SW-ALI was established by inhalating 4ml/Kg sea water. Sixty SD rats were randomly divided to two groups: normal control and sea water treatment group. At 1h、2h、4h and 8h behind inhalating sea water, PaO2、the wet/dry ratio of lung tissue、the pulmonary microvascular permeability,the MPO activity and level of MDA in lung tissue、the level of TNF-αand IL-1βin serum and BALF were examined.The expression of Shh、Ptc-1 and Gli1 was detected. The histological changes of lung tissue were observed under optical microscope.
6. Twenty SD rats were divided randomly four groups: control group、Shh treatment group、sea water treatment group、Shh+sea water treatment group. Rats in Shh and Shh+sea water treatment groups was jugular injection of recombinant human Shh at the dose of 6mg/kg. The rat model of SW-ALI was established ditto.PaO2、the wet/dry ratio of lung tissue、the pulmonary microvascular permeability、the expression of Ptc-1 and Gli1 were compared between the control group、Shh treatment group、sea water treatment group、Shh+sea water treatment group.
Results:
1. Histological sections showed that tissue pieces were scissored from periphery lung lobes accurately.Ⅷfactor related antigen and CD34 expression detected by immunofluorescence was positive,and BSI binding test was positive,too.Transmission electron microscope showed lots of protuberance on cell membrane and pinosomes in cytoplasm.
2. Compared with normal control group,the expression of Shh、Ptc-1and Gli1 in mRNA and proterin level in PMVEC decreased significantly in sea water treatment groups. Microscope showed PMVEC with sea water treatment crenulated, the distance between cells shortened. Transmission electron microscope showed protuberance on cell membrane increased, mitochondrion collapsed and crista missed, pinosomes in cytoplasm and heterochromatin in nucleus increased.
3. Shh increased significantly the proliferation of normal rat PMVEC.the effect of Shh shows a dose-dependent model.Cyclopamine can remarkably inhibit the proliferation of Shh100 group.
4. Compared with normal control group,the all group of sea water treatment has a lower proliferation of PMVEC,but the groups of Shh treatment has a higher proliferation than sea water treatment group.After sea water treatment, apoptosis of PMVEC dereased significantly compared with normal control group,but apoptosis in Shh treatment groups were lower than sea water treatment groups, apoptosis in Cyclopamine treatment group were higher than Shh treatment groups
5. Compared with normal control group, PaO2 decreased and W/D ratio, pulmonary microvascular permeability, MPO activity and the level of MDA, the level of TNF-αand IL-1βin sea water groups increased;the expression of Shh、Ptc-1and Gli1 in mRNA and proterin level in lung tissue of decreased significantly in sea water groups.
6. Compared with sea water group, PaO2 increased significantly and W/D ratio and pulmonary microvascular permeability decreased significantly in Shh+sea water treatment group. The expression of Ptc-1and Gli1 in mRNA and proterin level in the lung tissue of Shh+sea water treatment group was higher than sea water treatment group.
Conclusion:
1. The proliferation of rat PMVEC was inhibited and the apoptosis of rat PMVEC was promoted by the stimulation of sea water.
2. Recombinant human Shh can activate Hedgehog signalling to promote the proliferation of normal rat PMVEC and inhibit the apoptosis of normal rat PMVEC.
3. The expression of Hedgehog signalling decreased in PMVEC treated by sea water. Recombinant human Shh can activate Hedgehog signalling to promote the proliferation of PMVEC and inhibit the apoptosis of t PMVEC treated by sea water.
4. Rat model of SW-ALI was successfully established. The pulmonary microvascular permeability gradually increased and the pulmonary edema aggravated with the increase of time. The expression of Hedgehog signalling decreased in the lung tissue of SW-ALI rat model. The pulmonary microvascular permeability and pulmonary edema can be improved by recombinant human Shh in rat model of SW-ALI. This effect of Hedgehog signaling pathway may be mediated by the protective role on PMVEC.
海水淹溺不仅是航海事故、海上生产作业、旅游的死亡原因之一,而且是军队海上训练、作战、抢滩登陆战斗和非战斗减员的重要原因。海水淹溺后绝大部分病人都有肺损伤并继发肺水肿甚至海水型急性肺损伤(SW-ALI)/急性呼吸窘迫综合征(SW-RDS),使病情更加危重,救治极为困难。海水淹溺后高度肺水肿的形成与肺泡毛细血管膜的损伤密切相关,作为肺泡毛细血管膜的内屏障,肺微血管内皮细胞(pulmonary microvascular endothelial cell,PMVEC)的增殖与修复将有利于降低肺泡毛细血管膜的通透性,从而减轻肺水肿程度。因此研究肺微血管内皮细胞损伤后修复的信号转导机制,为防治海水淹溺ALI提供了新的思路。
Hedgehog(Hh)信号通路是与胚胎形成及形态发生有关的信号途径。主要由胞外的Hedgehog蛋白、细胞膜表面的特异性受体Patched (Ptc)及相关蛋白Smoothened (Smo)、胞内的转录因子Ci/Gli组成。Hedgehog蛋白是一种分泌型蛋白,在哺乳动物中包括Shh(Sonic hedgehog)、Ihh(Indian hedgehog)和Dhh(Desert hedgehog)三种家族成员。近年发现,Hh家族蛋白在许多成熟组织也有表达,调节细胞的增殖和自我更新,参与组织的损伤修复,并与多种疾病的发生发展相关。研究表明,Hedgehog (Hh)信号通路的激活可促进微血管内皮细胞增殖,诱导微血管重建。在此基础上,我们推测海水吸入ALI时,通过激活Hh信号通路,可促进肺微血管内皮细胞增殖,有利于肺泡毛细血管膜损伤后修复,从而减轻肺水肿程度。为此,本实验拟从细胞细胞水平检测Hh信号通路激活后对肺微血管内皮细胞增殖和凋亡的影响,从动物水平检测Hh信号通路激活后对肺微血管通透性和肺水肿程度的影响,为海水淹溺ALI的防治提供新的实验依据。
方法:
1.采用外周肺组织贴块法培养大鼠PMVEC,抽取组织块进行切片观察,以大鼠肺动脉平滑肌细胞和人脐静脉内皮细胞为对照,对培养细胞进行CD34、植物凝集素BSI、Ⅷ因子相关抗原免疫细胞化学染色鉴定,通过光镜和透射电镜观察细胞形态和超微结构。
2.将大鼠PMVEC随机分为正常对照组和海水处理2h、4h、8h和16h组。RT-PCR法分别检测细胞中Shh、Ptc-1和Gli1 mRNA表达情况;Western blotting法分别检测细胞中Shh、Ptch1和Gli1蛋白表达情况;光镜和透射电镜观察细胞形态和超微结构变化。
3.将大鼠PMVEC随机分为正常对照组和Shh50 ng/ml组、Shh100 ng/ml组、Shh200 ng/ml组、Shh400 ng/ml组和cyclopamine+Shh100 ng/ml组。MTT法检测各组细胞增殖活性。
4.大鼠PMVEC随机分为正常对照组、海水组、Shh50 ng/ml +海水组、Shh100 ng/ml +海水组、Shh200 ng/ml +海水组、Shh400 ng/ml +海水组和cyclopamine+Shh100 ng/ml+海水组。MTT法检测各组细胞增殖活性,TUNNL法检测细胞凋亡情况。5.采用屏气后经气管吸入4ml/Kg海水复制大鼠海水吸入ALI模型,随机分为对照组和海水组,每组又分为1h、2h、4h和8h四个时相点检测动脉血氧分压(PaO2)和肺组织湿干重比(W/D),肺微血管通透性、肺组织MPO活性和MDA含量、血清和BALF中TNF-α和IL-1β含量、RT-PCR法和Western Blot法分别检测肺组织Shh、Ptc-1和Gli1mRNA和蛋白表达水平,并进行肺组织病理形态学观察。
6.采用海水吸入ALI大鼠模型,静脉注射人重组Shh因子6mg/kg干预后,检测大鼠PaO2、肺W/D、肺微血管通透性、Ptc-1和Gli1mRNA和蛋白表达变化,并进行病理形态学观察。
结果:
1.组织块法培养大鼠PMVEC的综合鉴定结果显示:组织块源于外周肺组织,Ⅷ因子相关抗原和CD34免疫荧光化学染色阳性,植物凝集素BSI结合试验阳性,透射电镜细胞表面有较多突起,胞浆中有较多吞饮小泡。
2.与正常对照组比较,从4h开始,海水组细胞Shh、Ptc-1和Gli1 mRNA和蛋白表达量减少,8h最低,16h有所增加。光镜下可见海水处理16h后,PMVEC细胞皱缩,胞质减少,细胞间距增大,部分细胞脱落。电镜下可见海水处理16h大鼠PMVEC细胞表面突起减少,胞浆浓缩,线粒体水肿,嵴消失,胞浆中吞饮小泡增多,核内异染色质增多。
3. Shh处理正常大鼠PMVEC细胞增殖活性较对照组明显增强,并呈剂量依赖性。Cyclopamine+Shh100ng/ml组细胞增殖活性明显低于对应时相点Shh100ng/ml组。
4.与正常对照组相比,海水组PMVEC细胞增殖明显减少,而Shh处理各组细胞增殖活性明显高于海水组;Cyclopamine组细胞增殖活性明显低于Shh处理组。海水处理PMVEC 8h后,细胞凋亡较对照组明显增加,而Shh处理各组细胞凋亡明显低于海水组,Cyclopamine组细胞凋亡明显高于Shh处理组。
5.大鼠经气管吸入海水后,其PaO2明显低于对应时相点对照组,氧合指数<300;肺W/D、肺微血管通透性、肺组织MPO活性和MDA含量、血清和BALF中TNF-α和IL-1β含量明显高于对应时相点对照组,并随时间延长逐渐增加;肺组织Shh、Ptc-1和Gli1mRNA和蛋白表达水平显著低于对照组,并随时间延长逐渐降低。
6.海水吸入ALI大鼠注射人重组Shh因子后,其PaO2明显高于海水组,肺W/D和肺微血管通透性明显低于海水组,Ptc-1和Gli1mRNA和蛋白表达明显高于海水组。
结论:
1.海水刺激下大鼠PMVEC增殖受抑制,细胞凋亡增多。
2.人重组Shh可通过激活Hedgehog信号通路,促进正常大鼠PMVEC增殖并抑制其凋亡。
3.Hedgehog信号分子在海水处理大鼠PMVEC中表达降低,采用人重组Shh激活Hedgehog信号通路可促进海水处理大鼠PMVEC细胞增殖,抑制细胞凋亡。
4.成功建立海水吸入ALI大鼠模型。海水吸入ALI大鼠肺组织中肺微血管通透性和肺水肿程度随时间延长进行性增加;Hedgehog信号分子在海水吸入ALI大鼠肺组织中表达降低,采用人重组Shh激活Hedgehog信号通路后,可降低海水吸入ALI大鼠肺微血管通透性,减轻肺水肿程度。这种效应可能是Hedgehog信号通路激活后通过对PMVEC的保护作用实现的。
Backgroud and Objective:
sea water drowning is not only an important cause of death in the accident of civil sailing, wrecking on the sea, but also the key factor of army non-military depletion of numbers in training or fighting on the sea. As the consequence or result of sea water drowning, lung injury, secondary pulmonary edema or sea water respiratory distress syndrome(SW-RDS) occurs in most of patients, and leads to the challenge of treatment. Alveolus capillary barrier injure plays a key role in the process of severe pulmonary edema. The proliferation and repair of Pulmonary microvascular endothelial cells can improve the permeability of the barrier and alleviate the degree of pulmonary edema. Therefore, the understanding of signaling mechanism involved in PMVEC reparation after sea water drowning injure will provide new treatment strategies.
The Hedgehog signaling pathway plays a fundamental role in the development of various embryonic tissues development. It is composed of Hh protein、the transmembrane receptor protein patched (ptc) and smoothend(smo) and the transcription factor Gli. Hh is a protein secreted by cells. There are three kinds of it: Sonic Hedgehog(Shh)、Indian Hedgehog (Ihh) and Desert Hedgehog(Dhh).Recent investigations found that main component molecules of Hh pathway are present in many mature tissue and regulat cell proliferation and renewal in the process of repairation after injure. The activation of Hh signaling pathway can increase PMVEC proliferation and induce capillary remodeling. Thus we hypothesize that activation of Hh pathway can improve the PMVEC proliferation (survival) and reparation in the process of sea water drowning induced lung injure. Therefore we investigate the effect of Hh signaling pathway on proliferation and apoptosis of PMVEC, and the improvements of pulmonary microvascular permeability and pulmonary edema in rat sea water drowning model.
Methods:
1. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Histological sections from peripheral lung tissue pieces used for cell culture were examined. Used rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as control, CD34, lectin from Bandeiraea simplicifolia and factorⅧrelated antigen in the cultured cells were quantified by immunocytochemical staining. In addition, the cell morphology and ultrastructure were observed with inverted optical microscope and transmission electron microscope respectively.
2. PMVEC were divided randomly five groups:normal control, sea water treatment 2h、sea water treatment 4h、sea water treatment 8h and sea water treatment 16h. The expression of Shh and Ptc-1 mRNA was detected by RT-PCR and the protein expression by Western blot . The changes of cell morpha and ultrastructure were investigated with microscope or electron microscope.
3. PMVEC were divided randomly sis groups: normal control, Shh 50 ng/ml treatement group, Shh 100 ng/ml treatement group, Shh 400 ng/ml treatement group,and cyclopamine treatment group.MTT was used to detect the proliferational activity of PMVECs.
4. PMVEC were divided randomly seven groups: normal control, sea water treatment group、Shh 50 ng/ml+sea water treatment group、Shh 100 ng/ml+sea water treatment group、Shh 200 ng/ml+sea water treatment group、Shh 400 ng/ml+sea water treatment group,and cyclopamine +sea water treatment group. MTT was used to detect the proliferational activity of PMVEC.TUNNL was used to examinate the apoptosis of PMVECs.
5. A rat model of SW-ALI was established by inhalating 4ml/Kg sea water. Sixty SD rats were randomly divided to two groups: normal control and sea water treatment group. At 1h、2h、4h and 8h behind inhalating sea water, PaO2、the wet/dry ratio of lung tissue、the pulmonary microvascular permeability,the MPO activity and level of MDA in lung tissue、the level of TNF-αand IL-1βin serum and BALF were examined.The expression of Shh、Ptc-1 and Gli1 was detected. The histological changes of lung tissue were observed under optical microscope.
6. Twenty SD rats were divided randomly four groups: control group、Shh treatment group、sea water treatment group、Shh+sea water treatment group. Rats in Shh and Shh+sea water treatment groups was jugular injection of recombinant human Shh at the dose of 6mg/kg. The rat model of SW-ALI was established ditto.PaO2、the wet/dry ratio of lung tissue、the pulmonary microvascular permeability、the expression of Ptc-1 and Gli1 were compared between the control group、Shh treatment group、sea water treatment group、Shh+sea water treatment group.
Results:
1. Histological sections showed that tissue pieces were scissored from periphery lung lobes accurately.Ⅷfactor related antigen and CD34 expression detected by immunofluorescence was positive,and BSI binding test was positive,too.Transmission electron microscope showed lots of protuberance on cell membrane and pinosomes in cytoplasm.
2. Compared with normal control group,the expression of Shh、Ptc-1and Gli1 in mRNA and proterin level in PMVEC decreased significantly in sea water treatment groups. Microscope showed PMVEC with sea water treatment crenulated, the distance between cells shortened. Transmission electron microscope showed protuberance on cell membrane increased, mitochondrion collapsed and crista missed, pinosomes in cytoplasm and heterochromatin in nucleus increased.
3. Shh increased significantly the proliferation of normal rat PMVEC.the effect of Shh shows a dose-dependent model.Cyclopamine can remarkably inhibit the proliferation of Shh100 group.
4. Compared with normal control group,the all group of sea water treatment has a lower proliferation of PMVEC,but the groups of Shh treatment has a higher proliferation than sea water treatment group.After sea water treatment, apoptosis of PMVEC dereased significantly compared with normal control group,but apoptosis in Shh treatment groups were lower than sea water treatment groups, apoptosis in Cyclopamine treatment group were higher than Shh treatment groups
5. Compared with normal control group, PaO2 decreased and W/D ratio, pulmonary microvascular permeability, MPO activity and the level of MDA, the level of TNF-αand IL-1βin sea water groups increased;the expression of Shh、Ptc-1and Gli1 in mRNA and proterin level in lung tissue of decreased significantly in sea water groups.
6. Compared with sea water group, PaO2 increased significantly and W/D ratio and pulmonary microvascular permeability decreased significantly in Shh+sea water treatment group. The expression of Ptc-1and Gli1 in mRNA and proterin level in the lung tissue of Shh+sea water treatment group was higher than sea water treatment group.
Conclusion:
1. The proliferation of rat PMVEC was inhibited and the apoptosis of rat PMVEC was promoted by the stimulation of sea water.
2. Recombinant human Shh can activate Hedgehog signalling to promote the proliferation of normal rat PMVEC and inhibit the apoptosis of normal rat PMVEC.
3. The expression of Hedgehog signalling decreased in PMVEC treated by sea water. Recombinant human Shh can activate Hedgehog signalling to promote the proliferation of PMVEC and inhibit the apoptosis of t PMVEC treated by sea water.
4. Rat model of SW-ALI was successfully established. The pulmonary microvascular permeability gradually increased and the pulmonary edema aggravated with the increase of time. The expression of Hedgehog signalling decreased in the lung tissue of SW-ALI rat model. The pulmonary microvascular permeability and pulmonary edema can be improved by recombinant human Shh in rat model of SW-ALI. This effect of Hedgehog signaling pathway may be mediated by the protective role on PMVEC.
引文
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