The Study Of Signal Pathway In TGF-?1 Induced Fibrosis Of RMMCs

Background:Hydrocephalus(external hydrocephalus EH), is a common clinical brain disease, which seriously affects the quality of life of patients and becomes the heavy burden of the patients' family as well as the society. Once the hydrocephalus is formed, the rate of the disability is very high. The current clinical treatment of hydrocephalus can alleviate symptoms to some degree, but cannot cure the disease completely. Therefore, the treatment of hydrocephalus is a big problem in the Department of neurology. Common causes of the various hydrocephalus are subarachnoid adhesions, fiber hyperplasia Meningeal fibrosis is the main pathological change. The main cellular components of leptomeningeal for leptomeningeal cells are known as menigeal mesothelial cells,(MMCs). When there are the central nervous system infection and trauma cases, the meningeal mesothelial cells will proliferate and synthesize a variety of extracellular matrix, resulting in leptomeningeal fibrosis. Therefore, meningeal mesothelial cell culture mechanism research can provide an experimental model for leptomeningeal fibrosis and hydrocephalus formation, which is helpful for the understanding of the role and function of the meningeal mesothelial cells in the meninges fibrosis, and which is of great significance of the pathological mechanism of the development of meninges fibrosis occurrence.But the demanding work of primary cell isolation, culture and passage in vitro seriously limits the studies in this field.Current studies on meningeal fibrosis mainly focus on the imaging and histological level. The investigations of its pathogenesis at molecular level are lacking. Transforming growth factor-beta 1(TGF-?1) is one of critical fibrosis-inducing cytkines. Connective growth factor(CTGF), an important downstream factor that mediates the pro-fibrotic effects of TGF-?1, by promoting mitosis and fibroblast proliferationinducing collagen synthesis, and mediating cell adhesion and chemotaxis. Research results show that TGF-?1 is an important factor for the induction of meningeal fibrosis by inducing the m RNA and protein expression of CTGF in meningeal mesothelial cells, TGF-?1 promotes the formation of fibrosis, suggesting the significance of blocking TGF-?1 pathway in delaying the progression of meningeal fibrosis. However, TGF-?1 exerts many important physiological functions, including immune modulation and anti-inflammatory response, suggesting that blocking TGF-?1 function in a direct way is clinically unfeasible. Therefore, novel downstream targets of TGF-?1 need to be identified to specifically inhibit the pro-fibrosis effect of TGF-?1. Accumulated data showed that p38 and ERK/MAPK signaling pathway can mediate TGF-?1-induced fibrosis in various types of cells, such as lung fibroblasts, mesangial cells and renal fibroblasts. However, whether these signaling pathways are involved in TGF-?1-induced meningeal fibrosis remains unclear.The present study aimed at the establishment, identification and application of immortalized RMMCs,then we also established a cellular model of meningeal fibrosis with TGF-?1 as an inducer. The specific role of p38 MAPK and ERK/MAPK signaling pathway in TGF-?1-induced meningeal fibrosis of mesothelial cells was investigated, with the purpose to identify new effective intervention targets for the prevention and treatment of hydrocephalus. Objectives:1.To obtain the immortalized RMMCs as to develop an ideal cell model for the study of meningeal fibrosis in future.2.To establish a cellular model of meningeal fibrosis with TGF-?1 as an inducer and observed the role of p38 MAPK and ERK/MAPK.Researching the expression of P38 MAPK and ERK/MAPK, observing the expression of CTGF by the application of p38 MAPK inhibitor SB203580,or ERK/MAPK inhibitor U0126, so to explore the pathogenesis of meninges fibrosis. Methods:1. Primary rat menigeal mesothelial cells were isolated and cultured in vitro by mechanical trituration and light digestion method;2. Primary cultured RMMCs were infected by retrovirus mediated SV40 LT.3. Primary cultured and transfected RMMCs were identified by electron microscopy, immunocytochemistry and immunofluorescence chemical methods in morphology, specific marker expression and identification. We aimed to obtain the immortalized RMMCs and established an ideal cell model for the study of meningeal fibrosis.4.Incubating RMMCs with different concentrations of TGF-?1 24 h respectively, detecting the expression of CTGF m RNA by RT-PCR and the quanty of p-p38 MAPK ? p-ERK1/2 protein by Western blot; incubating with different concentrations of SB203580 and U0126 in advance, and then TGF- beta 1 was used to incubating the RMMCs 24 h, measured the expression of CTGF protein. Results:1.Primary RMMCs were successfully isolated. Isolation and culture of primary cells under inverted microscope was a polygon, the confluence of a cobblestone; scanning electron microscope(SEM) cell surface visible dense microvilli; immunofluorescence staining showed that the cultured cells were cytokeratin protein antigen positive; Immunocytochemistry results showed cultured cells were cytokeratin protein and vomiting were positive for factor VIII related antigen negative, training the purity of the cell reached more than 90%.2. The RMMCs were transformed by the lentvirus vector containing with SV40 LT into the HUVECs. The immortalized HUVECs shared the similar characteristics with the primary HUVECs, such as the cobblestone morphology,W-P body, Factor ?, KDR and the ability of tube formation.3. TGF-?1 can induce fibrosis of rat RLMC in vitro and RMMC can be used as an in vitro cellular model of meningeal fibrosis.4.TGF-?1 increased the phosphorylation of p38 in RLMC in a dose-dependent manner, indicating that TGF-?1 is able to activate p38 signaling pathway in RMMC.5.TGF-?1 increased the phosphorylation of ERK1/2 in RMMC in a dose-dependent manner. ERK/MAPK inhibitor U0126 didn't block the expression of CTGF. Conclusion:1. The immortalized RMMCs were successfully transformed by SV40 LT.2. TGF-?1 induces the expression of CTGF in meningeal mesothelial cells in a dose-dependent manner through p38 MAPK. It is suggested that p38 MAPK pathway is an important pathway through which TGF-?1 induces the expression of CTGF.3. TGF-?1 can induce the cells activation of ERK/MAPK, but the MAPK/ERK pathway is not participate in the process of meningeal fibrosis caused by TGF-?1.4. TGF-?1 may induces the expression of CTGF in meningeal mesothelial cells through a variety of ways.