Isolation,Culture, and Identification of Rats'adipose Derived Stem Cells (ADSCs) and Construction of Lentiviral Vector Carrying Rat Myocardin Gene
|School||Southern Medical University,|
|Keywords||Erectile dysfunction Diabetes mellitus Gene therapy Myocardin ADSCs Lentiviral vector|
BACKGROUNDErectile dysfunction (ED) is a kind of common disease of Andrology, the increase of age and basic diseases increased significantly higher risk of ED. Diabetes and ED have been proved to be highly related. Diabetes groups happen ED probability is3times of diabetic population, incidence of35%to90%. The pathogenesis of diabetic ED is a complicated process, So there has a great difficulty in the treatment, a significant portion of patients with diabetic ED is not sensitive to first-line drugs (PDEs), and unable or unwilling to accept second-line and third-line treatment. Thus, exploring some related factors basing on the abnormal pathophysiology will be a new idea for treatment of ED. Corpus cavernosum smooth muscle (CCSM) is the structural basis of cavernous space relaxing and penile erection, which plays a key role in the change of hemodynamics when the penis erects. Studies have shown that corpus cavernosum smooth muscle apoptosis increases and proliferation rate is reduced in diabetic, so the number of corpus cavernosum smooth muscle cells decreased significantly, cell ultrastructure pathological changes, decreased corpus cavernosum tissue compliance, trigger ED. Therefore, to increase the number of diabetes patients with corpus cavernosum smooth muscle cells and repair cells function and improve the function of corpus cavernosum tissues may be an important way to improve the level of prevention and cure of diabetic ED.Similar to vascular smooth muscle cells according to structure and function, corpus cavernosum smooth muscle(CCSM) cells can be further divided into two types:the contraction type and synthetic type. The major function of the former one is contraction, and that of the latter one is proliferation, migration, secretion and regulation of extracellular proteins, etc. The contraction type vascular SMC can transform into synthetic type ones and get to be proliferation when the vascular is injured or the vascular SMC cultured in vitro are stimulated by the growth factors. There is a structure of sinusoid in the corpus cavernosum, which is very similar with the vascular systems. Therefore, the corpus cavernosum is considered as a special kind of vascular tissue as well. Our previous study has demonstrated that the contraction type vascular SMC has transform into synthetic type ones in CCSM cells of diabetic rat with ED, cell diastolic function damage.Stem cells is a multi-directional differentiated potential and self-replicating features of undifferentiated cells collectively, according to its source can be divided into embryonic stem cells and adult stem cells. Adipose derived stem cells, ADSCs is a kind of adult stem cells, has multi-directional differentiated potential, under certain conditions can be induced differentiated into bone cells, cartilage cells, myocardial cells, fat cells, muscle cells and endothelial cells, etc. ADSCs have lots of advantages: draw materials easily, less tissue damage, etc. Therefore occupy a important position in regenerative medicine, can provide reliable source of cells for tissue engineering, lay a solid foundation for clinical application.Myocardin is a kind of the nuclear receptor transcription factor, play an important role in regulating smooth muscle cell differentiation process. Myocardin directly combine with serum effect factor (SRF), the combination of activation is regulated by the SRF series of scaffolds can be encoded protein and contraction of gene transcription.Recent studies have shown that Myocardin also play a important role in the ADSCs differentiation, Myocardin gene-modified ADSCs can differentiation into the smooth muscle cell. Our team hopes to further research:by transgenic methods, select suitable expression vector, guide Myocardin genes into ADSCs and as seed cells transplanted into rat corpus cavernosum, improve the extracellular environment and local tissue function of treatment area, develop ADSCs multi-directional differentiation potential, in effect of Myocardin differentiation into smooth muscle cells with diastolic function, by increasing the number of corpus cavernosum smooth muscle cells and repair diastolic function is expected to further improve diabetic ED rats penile erectile function.Before animal experiments, you first need to have mature cell culture techniques, which can obtain a large number of high purity, activity, suitable for gene transfection cell in a short term, then need to build stable Myocardin gene recombinant lentivirus vectors.According to the needs of research, this course summarized a set of relatively mature method of ADSCs isolation, cultivation and identification, and successfully build rat Myocardin gene recombinant lentivirus vectors, laid a solid foundation for further research work.OBJECTIVE1. Isolated ADSCs from adipose tissue of SD rats and through the in vitro observation its growth form, growth and growth curve, directional differentiation induction, related marker detection, identification, establish the cultivation of rats ADSCs model.2. Build rat Myocardin gene recombinant lentivirus vectors to lay a good foundation for subsequent work. METHODS1. Primary culture of rat ADSCs.A2-month-old SPF male rat, anesthesia the rat at first, take adipose tissue from epididymis, suture the cut, continue to raise animals. Remove blood vessels visible to the naked eye, fascia tissue, rinse with PBS, cut up, add2times the volume0.1%I-collagenase,37℃,60min,120R/MIN shocks digestion, use DMEM containing10%FBS, such as volume to termination digestion,1500r/min,8min, abandoned supernate, DMEM containing10%FBS suspension sediment, use100um cells sieve to remove the cell mass. Collect the filtrate, cell inoculation in25cm2culture bottle, in a standard environment culture. the first replacement medium after24h, remove adherent cells and the rest of the blood cells, every2～3d replace culture medium, under a microscope observation of cell morphology, growth, and take pictures.2. Subculture of rat ADSCs.Cell grow all over80%of the bottle bottom, with0.25%trypsin containing EDTA digestion, made cell suspension. Use count plate count cell suspension concentration, to adjust cell concentration, usually in the form of1x105～3×105/ml, vaccination in2～3new culture bottle or in a petri dish, namely according to1:3subculture.3. The growth curve of rat ADSCs by MTT method.Take grow full culture bottle bottom80%at third,sixth generations of ADSCs, digest centrifugal,1x103～1x104/hole density inoculated on96-well plates, after24h,take out3holes a day, MTT colorimetric,7days and replace culture medium every3d.. Time as the average transverse, OD as vertical axis to draw growth curve.4. The cryopreserved and recovery of rat ADSCsTake grow full culture bottle bottom80%at third generations of ADSCs, put it into cryopreserved tubes after digest centrifugal,5×105/ml, Freeze-stored liquid dispensing ratio is DMSO:FBS:basic culture medium=1:2:7.4℃for1h,-20℃placed2h,-80℃to place for the night, stored in liquid nitrogen. Recovery after1month, To determine survival, induced osteogenesis and fat cells experiment.5. Multi-directional differentiation potential detectionWith specific induction in vitro culture system to induce ADSCs into osteoblast and fat cells differentiation, and alizarin red staining and oil red O staining were used respectively to identify directional differentiation results.6. ADSCs surface markers detectionGrowth in good condition of third generation of ADSCs, made into single cell suspension, density of1x106/ml, join the rabbit anti rat CD29-PE, CD44-FITC monoclonal antibody working liquid100ul and set up control, flow cytometry instrument to detect cell surface markers, EXPO32. V1.2software analysis.7. The construction of recombinant lentiviral vector targeting rat MyocardinAdopts full length gene synthesis method of synthetic for the gene Myocardin in rats. Building and identification of recombinant plasmid pLVX-IRES-Neo-myocardin. Extract pLVX-IRES-Neo-myocardin plasmid, will carry the purpose gene in the rat rat myocardin slow virus expressing plasmid transfection and293t cells can packing the slow virus particles containing the purpose gene lenti-myocardin. Determination of virus particles Lenti-myocardin drops by rt-pcr and Western blot technique, testing rats myocardin gene expression of mRNA and protein levels.RESULTS1. The rat ADSCs cell morphological observationObserved under inverted microscope, primary generation of rat ADSCs4～6hours after inoculation began to stick wall, most were post wall and begin to stretch within24h, around7d, microscopically a lot into fiber cell growth and cell arrangement has a certain direction, about8d cells can be up to80%～90%single fusion, cells were fusiform after wedding, protrusions decreased, cell size and shape is consistent, primitive cells proliferate faster than fast, generally3～4d can form80%single fusion, still dark fibroblast cell growth, beam shaped and vortex pattern. Transfer after6generations, the cells are arranged fasciculation and swirling, still active hyperplasia, after10generation has, not seen obvious slow down cell proliferation rate, cell remain strong proliferation ability and morphological features uniform long shuttle samples, no cells in the process of the batches for spontaneous lipid droplets in cytoplasm.2. The growth curve of rat ADSCsThe growth curve of3,5,7generation rats ADSCs are in "S" shape, the ADSC growth curve as growth adaptation in1,2d,2d after cell growth accelerated, a logarithmic growth, after6d peak growth, cell proliferation rate drop, into the plateau.3. The cryopreserved and recovery of rat ADSCsRecovery the third generation of ADSCs1months after freeze, the determination of cell survival rate was79%, the recovery of cell growth form is good, after a long shuttle shaped fiber growth, develop to the3d wedding, proliferation force no decrease compared with cryopreserved before, into fat and osteogenetic differentiation also succeed.4. Multi-directional differentiation potential detection14days after induced osteogenesis, morphological changes were observed under inverted microscope visible most of the cells, much by short long shuttle deformation for spindle, oval and polygon cells, the nucleus gets bigger, it become more visible tiny black particles in cytoplasm, cell, granular nodular form sizes. For osteogenesis after induction of28d of the cells by alizarin red stain is visible:in central region colony growth of cells, extracellular matrix in black crumb phosphate precipitation, alizarin red staining the negative controls.7days after induced fat cells, inverted microscope is a small amount of cells within the cytoplasm of high refractive round small lipid droplets, cell morphological changes, from long spindle get round. Along with the induction of the extension of time, the increase in the number of cells gradually appear fat drops.14d after induction, increased cell volume, intracytoplasmic circular lipid drops, oil red O stain is visible:circular appear red particles inside the cell, the cytoplasm content of fat droplets. In the control group no significant change, is still the spindle cells grow into fiber samples, did not see of lipid droplets in cytoplasm, oil red O staining is negative.5. ADSCs surface markers detectionRat ADSCs CD29, CD44markers flow cytometry instrument detection, visible ADSCs CD29and CD44in the positive rate is high, the rate of positive of CD29, CD44were93.5%,87.2%.6. The construction of recombinant lentiviral vector targeting rat MyocardinResults show that rats myocardin protein successfully express in ADSCs, and illustrates the recombinant of lentiviral vector targeting rat Myocardin is successful.Conclusion1. Epididymal adipose tissue near based method based method is superior to the other parts, easy to get a lot of ADSCs, takes less susceptible to bacterial contamination, but many animals drawn at the same time;2. The separation experiment of rat ADSCs growth form is good, consistent with literature reports;3. Morphology, and stem cell surface markers directional differentiation induced experimental identification, detection and separation of cells of rat ADSCs; 4. Cell growth rate and morphology observation after the wedding the generation to come to the conclusion:3,5, or rat ADSCs proliferation rate of the seventh generation, so3-5generations of rat ADSCs suitable for use in large animal experiments;5. Successfully build rat Myocardin gene lentivirus vector.