| Background:Diabetic nephropathy(DN) is a major complication in patients with diabetes and represents the leading cause of end-stage renal disease. 25-40 percent of patients with type 1 and 2 diabetes will develop DN in 20-25 years. Currently, there is no cure for DN. Palliative therapeutic strategies include medical interventions for tight glycemic and hypertension control, dialysis and kidney transplantation could manage DN onset and progression. However, the curative effect was not satisfied due to the limitation of treatment methods.Bone marrow derived stroma cells(BMSCs) are well recognized for they are easily obtained from bone marrow with high amplification potential and involving no moral problems. Studies have shown MSCs, which possess multipotent differentiation characteristics, capacity for self-renewal and immune regulation abilities, are considered as a therapeutic agent to control DN progression and alleviate renal injury. However, many studies have reported that specific BMSCs homing and survival in kidney tissues at very low levels after intravenous transplantation, which has limited the effective implementation of BMSC-based therapies. Therefore, developing a method to improve BMSC homing to tissue impaired by DN is essential for clinical application.The underlying mechanisms by which intravenously injected BMSCs home to the site of damaged or inflamed parenchyma are not well understood, but their homing behavior pathology is thought to be analogous to leukocyte recruitment to a site of inflammation. Numerous studies have suggested that the release of some cytokines, adhesion molecules and trophic factors associated with inflammation and injury is of great importance for BMSC adhesion and the subsequent transmigration from the vasculature into inflamed tissues. Stromal cell-derived factor-1(SDF-1) and its unique receptor, CXCR4, play a critical role in BMSC homing and survival. However, cell cultureexpanded BMSCs do not express significant amounts of functional CXCR4 at the cell membrane; as a result of continuous passaging, they lose their migration and homing capabilities. Although viral vectors are effective, their high toxicity and high immunogenicity are associated with some serious side effects and the potential risk of oncogenic transformation. Pulsed focused ultrasound(p FUS) could improve BMSC therapies for targeted kidney tissues by establishing local chemoattractant gradients. However, FUS units are currently only approved by the US Food and Drug Administration for lower targeted homing ability. Therefore, developing an effective and non-invasive modality to improve CXCR-4 receptor expreaaion and trigger the release of chemoattractants within the local micro-environment is urgently needed to improve the therapeutic applications of BMSCs.Ultrasound-targeted microbubble destruction(UTMD) is a new, safe, non-invasive technology which has been used in increasing gene transfection efficiency, drug delivery efficiency and vascular permeability. Compared with viral vectors transfection technology, the combination of UTMD and non-viral is a safe and effective method to increase gene transfection efficiency. In addition, our group has shown that UTMD technology could significantly improve BMSCs targeted homing ability to ischemic myocardial areas and UUO kidney region. The kidney tissues are sensitive to biology effects for their high blood supply and high filtration. We wondered whether UTMDtechnology is effective or not in the application of stem cell therapy.Based on above findings, we attempt to explore whether combining UTMD technology and liposome will significantly increase CXCR-4 gene transfection efficiency in sub-cultured BMSCs. In addition, we wanted to explore whether microbubble-mediated diagnostic ultrasound irradiation on local kidney tissues could alter renal micro-environment by increasing the expression of adhesion factors, chemo-attract factors for increasing tem cell homing ability and alleviating DN progression. The study tested the feasibility, safety of increasing BMSCs homing ability by this therapy modality and effect for DN, and tried to explore the relevant mechanism, in order to provide a new way for increasing stem cell treatment efficiency.Objective:1. To study the efficiency, feasibility and mechanism of MB mediated therapeutic ultrasound combined liposome for increasing the transfection efficiency of CXCR-4 gene in sub-cultured BMSCs, and evaluate the migratory ability of transfected BMSCs in vitro. An attempt was made to find a non-viral method to achieve high transfection efficiency and improve migratory ability in vitro.2. To investigate the feasibility and safety of increased BMSCs homing ability to normal and DN rat’s kidneys induced by diagnostic ultrasound mediated microbubble destruction, and evaluate the possible mechanism.3. To study the efficiency, feasibility and the possible mechanisms of the increased homing ability of CXCR-4 modified BMSCs which was induced by diagnostic ultrasound mediated UTMD in order to explore experimental foundation for DN therapy.Methods:1. Transfection of CXCR-4 using microbubble-mediated ultrasound irradiation and liposomesWe used adherent culture method to isolate and cultivate BMSCs. The third generation BMSCs at logarithmic growth phase were chosen for the following experiment. Flow cytometry(FCM) was used to detect cell growth cycle and the expression of stem cell surface marker molecules. Osteogenic and adipogenes induction of BMSCs were performed. The plasmid DNA: p Ds Red-CXCR-4 was constructed according to gene BLAST results. Combing UTMD and liposome was applied to transfer gene in BMSCs. Laser scanning confocal microscope(LSCM), reverse transcription PCR(RT-PCR), western-blot(WB), FCM and immunofluorescence technology were applied to detect CXCR-4 receptor expression. Transwell migration assay was conducted to detect the migration ability of CXCR4-modified BMSCs. Scanning Electron Microscopy(SEM) was applied to assess the possible primary mechanism of increased gene delivery efficiency.2. Investigation into the impact and safety of diagnostic ultrasound mediated microbubble destruction on BMSCs homing ability to healthy rat’s kidneyRats were intravenously injected with red fluorescent protein(RFP)-labelled BMSCs which are transfected with lentivirus and then sonicated with microbubble-mediated ultrasound. The experimental rats were randomly divided into four groups:(1) Con group(BMSCs injection only);(2) MB group(MB was first injected, followed by BMSC injection);(3) US group(diagnostic ultrasound sonication followed by BMSC injection) and(4) UTMD group(UTMD following by BMSC injection). Four cohorts of rats were killed at 12 h, 24 h, 48 h and 72 h post-treatment. Their kidneys were rapidly dissected. LSCM was used to evaluate the number of BMSCs located in frozen kidney sections(5 mm in thickness). In addition, real-time PCR(RT-PCR) analysis was performed to investigate RFP m RNA expression in rat kidney tissues, which could reflect the number of RFP-labeled cells located in kidney tissues. The levels of blood urea nitrogen(BUN) and serum creatinine(Scr) were measured to evaluate renal function analysis. Haematoxylin and eosin(H&E) and periodic acid–Schiff(PAS) for histopathologic examination were performed to assess the safety. WB and ELISA were assayed to evaluate the expression changes of SDF-1, VCAM-1, E-selectin and VEGF and cytokines in kidneys. To assess the possible mechanisms for kidney microenvironment changes, we investigated ultra-morphology changes of kidney vascular endothelial cells using transmission electron microscopy(TEM).3. Enhanced homing ability and retention of BMSCs to diabetic nephropathy by microbubble mediated diagnostic ultrasound irradiationAll rats were received a single intraperitoneal injection of streptozotocin(STZ, Sigma), diabetic rats presented mild microalbuminuria(an early sign of DN) and were considered to be diabetic nephropathy. Only the right kidney was irradiated; the left kidney served as an internal control. RFP–labeled BMSCs were administered combined with microbubbles to DN rats. We observed enhanced BMSC homing and retention in microbubble-mediated diagnostic ultrasound-irradiated kidneys compared with the contralateral kidneys on days 1 and 3 post-treatment. RT-PCR analysis was performed to investigate RFP m RNA expression, which could reflect the number of RFP-labeled cells located in kidney tissues. The levels of BUN and Scr were measured to evaluate renal function analysis. H&E and PAS for histopathologic examination were performed to assess the safety. WB and ELISA were assayed to evaluate the expression changes of SDF-1, VCAM-1, E-selectin and VEGF and cytokines in kidneys. To assess the possible mechanisms for kidney microenvironment changes, we investigated ultra-morphology changes of kidney vascular endothelial cells using TEM.4. MB mediated diagnostic ultrasound sonication enhanced CXCR-4 modified BMSCs homing ability and improve DN outcomesThe experimental DN rats were randomly divided into six groups:(1) Con group;(2) UTMD group;(3) BMSCs group;(4) CXCR-4+BMSCs group;(5) UTMD+BMSCs group;(6) UTMD+CXCR-4+BMSCs. RFP–labeled BMSCs were administered combined with microbubbles to DN rats via tail vein. We observed enhanced BMSC homing and retention in microbubble-mediated diagnostic ultrasound-irradiated kidneys on 24 h, 48 h and 72 h post-treatment. RT-PCR analysis was performed to investigate RFP m RNA expression, which could reflect the number of RFP-labeled cells located in kidney tissues. We evaluate changes of blood glucose concentration, 24 h urinary protein and UAER value in DN rats on 3d, 7d and 14 d post-treatment. PAS staining was conducted to assess the possible renal pathology changes. Real time-q PCR and WB methods were assayed to evaluate the expression of TGF-β1 and TNF-αat each timepoint.Results:1. Rat BMSCs were successfully isolated and cultured in vitro. FCM results showed that specific antigens CD44, CD29, and CD90 were positively expressed and the specific antigens CD34, CD45, and CD11 b of other cell lineage were seldom expressed in sub-cultured BMSC. Adipogenic and osteocyte differentiation were successfully induced. Microbubble-mediated ultrasound irradiation and liposome was applied to transfer p Ds Red-CXCR-4 gene in vitro. FCM and LSCM results showed RFP protein expression was higher after treatment. WB and FCM results showed similar results as above. Transwell migration assay found that the migration ability of CXCR4-modified BMSCs was 9-fold higher than controls. Following microbubble-mediated ultrasound irradiation, SEM showed altered BMSC shape and tiny perforations on the cell membrane. The transfected BMSCs still maintained their differentiation and proliferation capability.2. BMSCs transfected with lentiviral vectors carrying RFP showed bright red fluorescence under LSCM. FCM showed that the level of RFP expression in BMSCs was approximately 97%. Quantification analysis of RFP-labelled BMSCs in renal tissue showed that the number of engrafted RFP-labelled BMSCs in the UTMD group was statistically greater than in the US group, MB group and Con group 12 h, 24 h, 48 h and 72 h post-treatment. HE staining showed no signs of cytolysis or cell necrosis following UTMD treatment; only mild haemorrhage and inflammation were observed after UTMD treatment. PAS staining demonstrated no obvious signs of renal tissue disorganization after UTMD treatment. There were no significant differences in the serum BUN and Scr levels at 12, 24, 48 and 72 h post-UTMD treatment compared to their respective pretreatment values. WB and ELISA results showed that the mechanical effects of UTMD would increase local and transient levels of chemoattractants(i.e. cytokines, integrins and growth factors) in targeted kidney tissues. Transmission electron microscopy showed that vascular endothelial cell was discontinuous in the UTMD group post-treatment, becoming smooth 72 h later.3. Three days after intraperitoneal injection of STZ, blood glucose maintained at a high level of more than 16.7 mmol/L. The DM rat model was established when the rats exhibited typical symptoms of polyphagia, polydipsia, urorrhagia and weight loss. Four weeks later, when the urinary micro albumin excretion rate was detected, early DN model was established. LSCM and Real-time q PCR showed enhanced BMSC homing and retention in microbubble-mediated diagnostic ultrasound-irradiated kidneys compared with the contralateral kidneys on days 1 and 3 post-treatment. The results from immunohistochemical analysis, Western blot and enzyme-linked immunosorbent assay indicated that the local and transient expression of various chemo-attractants(i.e., cytokines, integrins and trophic factors) found to promote BMSC homing was much higher than observed in nontreated kidneys. The local capillary endothelium rupture observed by transmission electron microscopy may account for local micro-environment changes. Histopathologic analysis revealed no signs of kidney damage.4. LSCM and Real-time q PCR showed more BMSC homing and retention in UTMD+CXCR-4+BMSCs group than other groups on 24 h, 48 h and 72 h post-treatment. Blood glucose of DN rats without any treatment increased progressively. After BMSC engraftment, blood glucose decreased significantly, but without correlation with stem cell amount. 24 h urine protein and UAER values were found decreasing significantly in BMSCs group, CXCR-4+BMSCs group, UTMD+BMSCs group and UTMD+CXCR-4+BMSCs group than Con group. Among all these groups, UTMD+CXCR-4+BMSCs group showed the least values. PAS staining demonstrated enlarged extra cellular matrix, glomerular sclerosis, mesangial proliferation and basement membrane thickening in untreated kidneys. Real-time q PCR and WB results showed the expression of TGF-β1 and TNF-αwas strong in Con group and UTMD group, and progressively increased with time going. As expected, the expression did not significantly increase in BMSCs treated groups. The expression of TGF-β1 and TNF-α in UTMD+CXCR-4+BMSCs group was the lowest among all groups involved.Conclusions:1. BMSCs were successfully obtained, cultured, identified and purified in vitro, showing low differentiation, high self-renewal ability and multi-potent differentiation.2. The combination of microbubble-mediated ultrasound irradiation and liposomes was proven useful to enhance p Ds Red-CXCR-4 gene transfection efficiency in third passage BMSCs and improve their migratory ability in vitro.3. RFP labeled BMSCs by lentvirus was bright and consistent in vivo, thus could efficiently track the distribution of BMSCs in organs.4. Renal microenvironment changes caused by appropriate UTMD may promote BMSC homing ability toward treated kidney tissues without renal toxicity and cell damage.5. Early DN rat model can be established by receiving a single intraperitoneal injection of streptozotocin and confirmed by random blood glucose concentration and 24 h urinary albumin.6. Renal micro-environment changes caused by appropriate microbubble-mediated diagnosticultrasound irradiation may promote BMSC homing ability to the diabetic kidney without renal toxicity and celldamage, which are activated by acoustic cavitation within a relatively short time window.7. The homing ability of CXCR-4 modified BMSCs to DN kidney tissues could be increased by MB mediated diagnostic ultrasound irradiation. Renal function was improved by inhibiting the expression of TGF-β1and TNF-α. |
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