A Study On The Ucmsc Derived Membrane Microparticles For Promoting The Is Chemia Limbs Angiogenesis

The animal test and clinical studies prove that mesenchymal stem cells (MSC) is effective to treat ischemic diseases. Currently, it is widely believed that the therapeutic effect of MSC is affected by its paracrine secretion of various soluble bioactive molecules. Since microparticles (MPs) can be released through eukaryotic cell apoptosis, it is used as the carriers of biological signals and information exchange between cells to transfer the biologically active substances and surface receptors to the selected target cells and thus adjust the biological function of target cells.In this test, the primary UCMSC after isolation and culture is identified in terms of the cell morphology, differentiation function and phenotype, and then UCMSC apoptosis is induced under the hypoxia and low-nutrition condition (simulating the apoptosis of MSC occurred during treatment of ischemic diseases) to obtain MPs from the conditioned medium. The rat limb ischemia model is taken as the research object, and the influence of MPs on angiogenesis of the ischemic area is observed. Finally, based on the study object of the human umbilical vein endothelial cells (HUVEC), and the influence of MPs on its proliferation, migration and the formation of tubular structures the present study preliminarily explores the mechanism of MPs for promoting angiogenesis.Adipogenic and osteogenic differentiation are carried out after isolation and culture of UCMSC; then CD29, CD31and CD44, CD45, CD73and other antigen markers of cell membranes are used to identify the cell phenotype by Flow Cytometry (FCM). Then through induction of UCMSC apoptosis in a hypoxia and low-nutrition culture condition and detection of the apoptosis rate at different times (Id,2d,3d,4d) by FCM, the culture conditions, which is manifested by a high apoptosis rate at the early period and a low apoptosis rate at the later period is obtained. Briefly, cell supernatants are obtained after additional centrifugation at100,000g for1hour at4℃to prepare the MPs. Annexin V-FITC binding and phenotypic profile are determined by FCM. MPs are observed directly under a transmission electron microscope. And the protein concentration is measured by BCA method, so as to establish the test method for MPs preparation. Secondly, the limb ischemia model is established by ligation of the right femoral artery in rats. Then the influence of MPs on the limb ischemia angiogenesis is detected with UCMSC as positive control and Buffer as negative control. Three groups of liquid is inoculated to the ischemic region on the following day after the establishment of the model.14days later, laser Doppler perfusion imaging (LDPI) test is carried out to test the blood supply of tissues. The muscle tissue paraffin sections are made, and the muscle tissue atrophy condition was evaluated through H&E staining sections; the skeletal muscle microvessel density is tested by the CD31immunohistochemical staining. Finally, with the study object of HUVEC, the MPs carrying Dil fluorescence is prepared with Dil marker on UCMSC and co-cultured with HUVEC, and then the infection rate of HUVEC is tested at different times so as to define the way of functioning MPs and HUVEC. To explore the mechanism of MPs angiogenesis, the test uses the DMEM,10%FBS, lOng-mL"1bFGF as control, and evaluates the influence of MPs on in vitro HUVEC proliferation, migration and ability of tubular formation by means of the flow cytometry detection of test methods of cell proliferation index, Transwell migration and formation of tubular structures in vitro.The primary UCMSC is successfully isolated and cultured. Its adherent cells are visible as fibrous form, which can be differentiated to osteogenic and adipogenic cells. In addition, CD29, CD44and CD73of positive expression and CD31, CD45of negative expression conformed to the MSC’s minimμm standards stipulated in the first international congress on controversies on placenta-derived stem cell; The longer the culture time of UCMSC under the hypoxia and low-nutrition condition is, the more the early apoptosis cells become; but on the fourth day, the late apoptosis is significantly increased. To avoid the cell necrosis and release of apoptotic bodies to contaminate MPs, the conditioned medium under three days of culture is used to prepare MPs, which is about0.15μm determined by flow cytometer identification, and its surface marker is consistent with the expression of parental cells. Moreover, the Annexin V expression is positive, The transmission electron microscopy to detect their diameters are around80-150nm, and consistent with the basic definition of MPs. The protein concentration is measured by BCA method, and it is about lmg·mL-1. After ligation of the right femoral artery in rats, LDPI detection is conducted and the results show that the blood flow perfusion on the right side is lowered by56.92%in contrast to the left side; In the ischemic area, on day14after inoculated by Buffer, MSC and MPs, the blood flow recovery of the right ischemic limb of the MPs group was significantly superior to that of the Buffer group and MSC group; the H&E staining of pathological specimens showed that the muscle cell atrophy was obvious in the Buffer group, and infiltration of nμmerous inflammatory cells can be observed. Muscle cells of the MPs and MSC group are tightly arranged as those of the normal group, and no obvious infiltration of inflammatory cells can be observed; the muscle capillary density in the ischemic areas is detected using CD31immunohistochemical staining, and the results showed that the muscle capillary density in the MP group is significantly higher than that of the Buffer group and normal group (P<0.01), slightly higher than that of the MSC group, but without statistical difference. The infection rate of DiI on UCMSC obtained through flow cytometry detection is about99%; when the MPs carrying Dil is prepared and cultured with HUVEC, after4hours and12hours, its infection rate of HUVEC is about 46.64%and86.55%; In the MP group, its proliferation promoting effect on HUVEC is significantly better than the other groups (P<0.01), and its ability of HUVEC migration and formation of tubular structure was significantly superior to DMEM and FBS group (P<0.01), slightly higher than the bFGF group, but there was no statistical significance.The above results show that the UCMSC consistent with MSC standard can be obtained from human umbilical cord; and the MSC-MP consistent with the definition of MPs can be obtained through apoptosis induced by the hypoxia and low-nutrition condition. The MPs prepared by above method has the same ability as MSC transplantation to promote ischemic limb angiogenesis and tissue repairing, which provides an evidence for promoting HUVEC proliferation and migration through MSC-MP, and formation of tubular structure. It is expected to explore a new therapeutic mechanism for the MSC, i.e., transfering from the paracrine mechanism to non-cell MPs mechanism.