Study Of Bone Marrow Stromal Cells To Treat Lymphedema And The Effect Of Percutaneous Renal Artery Angioplasty And/or Stenting (RA-PTAS) On Blood Pressure And Kidney Function

Part Ⅰ:Study of Bone Marrow Stromal Cells to Treat Lymphedema Background:Lymphedema refers to certain parts of the body, caused by the swelling of the tissues and organs of the lymphatic drainage barriers. In1983, the International Society of lymphatic President Casley-Smith JR is estimated that there are about140million people worldwide suffer from various types of lymphedema, which is about45million limb lymphedema, and the number is increasing. Unfortunately, the lymphedema from domestic to international research did not arouse the attention of the medical profession, WHO has not released the number of authoritative specialized lymphedema disease incidence statistics, data distribution, treatment, relative to other diseases study. However, we can be obtained some information from other diseases.Before1994, China’s lymphedema patients with elephantiasis is caused by filariasis. Zhang Disheng,is an Academy of Chinese Engineering Academy, say that the National elephantiasis of the number of patients is more than five million. In addition, upper extremity lymphedema patients required surgical treatment caused by breast cancer surgery is increasing of at least10,000people.According to the World Health Organization International Cancer Research Center released GLOBOCAN2008,2003-2007,32tumor registries point’s data show that Chinese breast cancer incidence is29.18/10million, new cases is about38million people. According to the2008data show that4%-56%of patients occurred upper limb lymphedema. So only the number of breast cancer lymphedema patients is increasing of15-210thousand. So far, there is no good method for the treatment of lymphedema. in the clinical, still use surgical, physical massage, electric and microwave, drugs treatment, but these methods are not satisfactory, the effect is short-lived, high recurrence rate or complications, it can not cure lymphedema.Bone marrow stromal cells is the adult bone marrow cells, except hematopoietic stem cells, and more able or totipotent stem cells, derived from the mesoderm. In1968, Friendenstein first isolated it from the bone marrow. Specific in vivo microenvironment induced differentiation of a variety of tissue cells, its main advantage is:1. Marrow stromal cells has a strong proliferation capacity and multi-directional differentiation potential;2. easy separation, training, amplification, purification, there is no immune rejection characteristics, repeatedly passaged amplification and after cryopreservation still has the characteristics of stem cells;3. fewer ethical issues;4. can transfer exogenous gene and efficient and stable expression. Therefore, have broad space for development in the study of regeneration of tissues and organs.In1996, vascular endothelial growth factor C, Swedish Joukov et al isolated it for the first time. Its gene is located on human chromosome4q34, molecular weight about46.9KD. Joukov and other studies suggest that it is a member of the VEGF family, and named the VEGF-C, VEGF-C receptor FLT-4(VEGFR-3). Messenger RNA of the VEGF-C is expressed in almost the entire human tissue, including embryonic. Into the body tissue in the heart, placenta, ovarian cancer, and small intestine of VEGF-C is highly expressed. The progress of the study of the VEGF-C is fast. Many studies have documented and elaborated its genetic makeup, the microscopic structure of the product as well as the biological function and transcriptional expression has also been a lot of reports. Currently hot research also focused on receptor expression regulation, etc. With further research, the researchers found that the expression of VEGF-C and lymphatic endothelial marker very closely linked to its ability to promote lymphatic endothelial cell proliferation, regulating its biological activity. Many studies show that it has two receptors, VEGFR-2and VEGFR-3. Combination with VEGFR-2can induce angiogenesis, combined with VEGFR-3can promote lymphangiogenesis. VEGF-C152S is a point mutant generated by the replacement of the second conserved Cys residue of the recombinant processed VEGF-C by a Ser residue. VEGF-C152S is analog to the human VEGF-C156S mutant and only active toward VEGFR-3/FLT-4but, unlike wild type VEGF-C, is unable to bind to and to activate signaling through VEGFR-2/KDR.Bone marrow stromal cells in the treatment of lymphedema is still in the initial stage, therefore, in order to obtain the VEGF-C152S how to regulation of rat lymphatic endothelial cells and lymphangiogenesis, using VEGF-C152S to induce bone marrow stromal cells. And use RT-PCR, Western blot and immunofluorescence techniques to observe. And then use removing a part of the skin and clean the deep lymphatic vessels and lymph nodes to make a secondary lymphedema model, and transplanted BMSCs into the model, observed treatment effect.Objective:This study was designed to investigate VEGF-C152S induced bone marrow stromal cells into the lymphatic endothelial cells, and the treatment to lymphedema.Methods:1. Obtain, separate, culture, amplify, and identify the rat bone marrow stromal cells in vitro.2. Take the third to five generations of cells using recombinant rat VEGF-A and VEGF-C152S in vitro to differentiate the rat bone marrow stromal cells, and determine the cells using RT-PCR, Western blot and immunofluorescence techniques, stem cells express the lymphatic endothelial cell-specific flag material Prox-1and LYVE-1, as well as vascular endothelial cell markers of CD34and of CD31, to observe the VEGF-A and of VEGF-C152S induced rat bone marrow stromal cell differentiate to the lymphatic vessels.3. Make the rat secondary lymphedema model, VEGF-C152S-induced bone marrow stromal cells were injected into the subcutaneous tissue of the rat model, to observe the therapeutic effect of lymphedema.Results:1. Obtain, separate, culture and identify the rat bone marrow stromal cells in vitro successfully2. Using recombinant rat VEGF-A and VEGF-C152S in vitro to differentiate the rat bone marrow stromal cells, and determine the cells using RT-PCR, Western blot and immunofluorescence techniques, we find that stromal cells express the lymphatic endothelial cell-specific flag material Prox-1and LYVE-1, do not express vascular endothelial cell markers of CD34and of CD31, we observed that the VEGF-A and of VEGF-C152S induced rat bone marrow stromal cell differentiate to the lymphatic vessels.3. We use an new method to make the lymphedema model based on the old method, it has a higher rate. We use the rat recombinant VEGF-C152S-induced rat bone marrow stromal stem cells to transplant in the model and found that it can increase lymphangiogenesis, and can treat lymphedema.Conclusion:1. The method is feasible to obtain, separate, culture, amplify the rat bone marrow stromal cells in vitro, using recombinant rat VEGF-A and VEGF-C152S can differentiate the rat bone marrow stromal cells towards lymphatic endothelial cell in vitro.2. Our new mehod "Circumcision after the surgery of cut the muscle and skin" create a good model of rat limb lymphedema, it has a higher rate.3. In rat limb lymphedema model, we use the rat recombinant VEGF-C152S-induced rat bone marrow stromal stem cells to transplant and found that it can increase lymphangiogenesis, and can treat lymphedema. And its therapeutic effect is related to the amount of cells, both5×10Λ6/per dose and1×10Λ6/per dose has effection,5×10Λ6/per dose is more pronounced. Bring New Ideas:1. According to our results induce cell differentiation, we first proposed the mechanism of VEGF-A to promote lymphangiogenesis is that it through the VEGFR-2signaling pathway to promote VEGFR-2high expression and cause Prox-1 upregulated, and then cause the cells express lymphatic endothelial cells markers. This study can provide new ideas and theoretical support for inhibiting tumor lymphangiogenesis and the treatment of lymphatic deficiency diseases.2. Our new mehod "Circumcision after the surgery of cut the muscle and skin" create a good model of rat limb lymphedema, it has a higher rate.3. We use the rat recombinant VEGF-C152S-induced rat bone marrow stromal stem cells to transplant it in rat limb lymphedema model and found that it can increase lymphangiogenesis for the first time,and find it can treat lymphedema. And its therapeutic effect is related to the amount of cells, both5×10Λ6/per dose and1×10Λ6/per dose has effection,5×10Λ6/per dose is more pronounced. all of those provide new ideas and experimental work for the treatment of human secondary lymphedema.Part Ⅱ:The Effect of Percutaneous Renal Artery Angioplasty and/or Stenting (RA-PTAS) on Blood Pressure and Kidney Function Background:Renal artery stenosis (RAS) can cause renovascular hypertension and ischemic nephropathy, frequently resulting in end-stage renal failure. Several studies reported increased prevalence of ischemic nephropathy with special reference to renal artery stenosis due to atherosclerosis in elderly patients. Hypertension due to RAS is sometimes not responsive to oral antihypertensive drugs. Instead of the traditional surgical approach, endovascular treatment has recently been accepted as the primary line of treatment of renal artery stenosis to revascularize the stenotic artery and prevent chronic renal insufficiency. However, whether this procedure can effectively controlling blood pressure and improve renal function remains controversial. Currently, there is no clear evidence that this procedure can prevent progressive deterioration of renal function.Nevertheless, there are patients with satisfactory outcomes in terms of improvement or stabilization of renal function. while a portion of patients may have deteriorated renal function after balloon angioplasty and stent deployment. In this study, we report on endovascular treatment of115patients with RAS who underwent endovascular treatment and evaluate the safety and efficacy of this procedure. Our experience confirm the efficacy of this procedure and provide helpful experiences for clinicians.Objective:To evaluate the clinical effect of endovascular treatment on postoperative blood pressure control and kidney function of hypertensive patients with renal artery stenosis (RAS).Methods:Between January2004and December2011, RAS was diagnosed in120renal arteries from115hypertensive patients. Pre-and post-operative blood pressure and glomerular filtration rate (GFR) were monitored. Postoperative oral antiplatelet and antihypertensive agents were administered. Clinical follow-up was available for all patients for at least6months.Results:Balloon angioplasty was performed successfully in110patients, and stents were deployed in94renal arteries from89patients. Hypertension was cured or lessened in19and61patients, respectively. Blood pressure was stable and worsened in26and9patients, respectively. The renal function improved and was stable in23patients and57patients, respectively. Deterioration of renal function was observed in11patients. Doppler ultrasound after discharge revealed87patent renal arteries and fixed stents in82patients6months after procedure.Conclusion:Balloon angioplasty and stent deployment is an effective and feasible procedure for patients with RAS that help in controlling blood pressure and improving renal function moderately. Bring New Ideas:Balloon angioplasty and stent deployment is an effective and feasible procedure for patients with RAS that help in controlling blood pressure and improving renal function.