Study On The Effect Of Lentiviral Vector-mediated EGFP Gene Modified Adipose-derived Mesenchymal Stem Cells Treating Cisplatin-induced Acute Kidney Injury Of Mice

Background Cisplatin is a kind of drugs widely used in the treatment of cancer. However, due to the adverse side effects of cisplatin, especially its nephrotoxicity, cisplatin clinical application is severely restricted. Since cisplatin has multiple molecular targets, the inhibition of a single target only plays a partial protection of nephrotoxicity. Therefore, the treatment of cisplatin-induced renal injury needs a new strategy. Compared to drug therapy of AKI, stem cell-based therapy posseses many potential advantages, such as the secretion of various cytokines, fusion with the host cell, activation of endogenous stem cells and differentiation within the injured cells, and so on. There are amount of mesenchymal stem cells within adipose tissue. Adipose mesenchymal stem cells have many advantages, for example, they are easily isolated, cultured, and expanded steadily in vitro, low immunogenicity,without ethical problems. In recent years, mesenchymal stem cells have investigated for the treatment of AKI, but the treatment mechanisms involved remain controversial.Objectives1. To screen the optimal multiplicity of infection (MOI) which LV transfectes ADMSCs and dectect the proliferation of transfected ADMSCs.2. To explore the effects and propose mechanisms of allogenic ADMSCs for the treatment of acute kidney injury induced by cisplatin to provide evidences of the clinical transplantation and scientific experiments with ADMSCs.Methods 1. ADMSCs of KM mice were isolated, cultured and identified in vitro. Surface markers of ADMSCs were detected by flowcytometry (FCM) and potential of multi-directional differentiation were detected by alizarin red staining and oil red O staining.2. The ADMSCs were transfected by lentiviral vector containing enhanced green fluorescent protein (EGFP) with different multiplicity of infection. FCM and MTT colorimetric assay were used to detect the transfection efficiency of LV transfecting ADMSCs and the vitality of transfected ADMSCs respectively.3.60male mice were randomly divided into normal control (NC) group, physiological saline (PS) group and ADMSCs treatment (AT) group with20mice in each group. ADMSCs were injected into the transplantation group through caudal vein after12h of cisplatin injection4. The serum was collected from heart blood after injected cisplatin for7days. The serum BUN and Scr levels were detected to evaluate the renal function; pathological changes were observed according to HE staining; apoptosis of renal epithelial cells were detected by the TUNEL method; aoptosis and expressions of Bcl-2and Bax protein, and proliferation and expression of ki-67protein in kidney were dectected with immunohistochemical staining; distribution of ADMSCs in vivo after transplantation was observed by fluorescence microscope; The levels of tumor necrosis factor-a (TNF-a), monocyte chemoactant protein-1(MCP-1), interleukin-1β(IL-1β), interleukin-10(IL-10), hepatocyte growth factor (HGF), bone morphogenetic protein-7(BMP-7) in renal tissue was detected by enzyme-linked immunosorbent assay (ELISA).Results1. ADMSCs were positive for CD29, but negative for CD34. Moreover they were identified pluripotent by differentiation in vitro. 2. Transfection efficiency was68.67%after infection for72h under optimal MOI (MOI-25). The proliferation of transfected ADMSCs had no significant alteration in1-7d of after infection for72h at MOI=25.3. The serum levels of BUN, Scr and acute tubular necrosis (ATN) score of AT group were significantly lower than those of PS group (P<0.05); Cell apoptotic index (AI) of renal tubular epithelial cells in AT group was lower than those in PS group, while proliferating cell nuclear antigen index (PI) was more than those in PS Group (P<0.05); The expression of Bcl-2, Bax in AT group were more than those in PS group (P<0.05), but Bcl-2/Bax ratio was higher than those in PS group (P<0.05). A few scattered cells expressing green fluorescent protein were dectected in kidneys of AT group by fluorescence microscope, however, ADMSCs were not trapped in kindneys of other groups. ELISA results showed that the expressions of pro-inflammatory cytokicines such as TNF-a, MCP-1and IL-1β in renal tissue of PS group were increased remarkably (P<0.05), while the expressions of anti-inflammatory cytokicines such as BMP-7and HGF decreased significantly compared to AT group (P<0.05), however, expression of IL-10was no statistically significant reduction compared to AT group (P>0.05).Conclusions1. Differential attachment technique is convenient and high-performance method for the separation of ADMSCs, and can become a new source to isolate cells for tissue regeneration.2. Lentiviral vectors carrying EGFP can effectively infect ADMSCs. The proliferation of transfected ADMSCs had no significant changes.3. Allogenic ADMSCs transplantation can accelerate proliferation and suppress apeptosis of renal tubular epithelial cell through down-regulating the expression of Bax protein and up-regulating the ratio of Bcl-2/Bax in acute kindey injury. 4. The transplanted ADMSCs can home to the injured kidneys and adjust the pro-inflammatory cascade by a paracrine manner, which subsequently promote tissue remodeling and protect the kidney from further injurys.