Primary culture and identification of rat skeletal muscle-derived stem cells / 中国组织工程研究

BACKGROUND: Skeletal muscle-derived stem cells (MDSCs), another adult pluripotent stem cells, have become a hot topic in the field of gene therapy and cell-based tissue engineering. It has wide prospect in treating stress incontinence by periurethral injection of patients' MDSCs.OBJECTIVE: To investigate the method of culturing MDSCs in vitro so as to provide experimental basis for treating stress incotinence by injecting MDSCs.DESIGN: Repeated observation.SETTING: Department of Reproductive Medical Center and Department of Urology, Renmin Hospital of Wuhan University.MATERIALS: This experiment was conducted at the Laboratory of Department of Urology , Renmin Hospital , wuhan University from December 2003 to May 2004. Totally 20 female SD rats , aged 4 to 6 weeks , were involved . Type- Ⅺ pancreatin, collgenase , pancreatin ,polylysine (Sigma), dispase enzyme (Gibco) , chick embryo extract (self made).METHODS: ① Rats were anesthetized intraperitoneally with 10 g/Lpentobarbital sodium (30 g/kg). Under aspetic condition, gastroeneminus were isolated and immediately put into pre-cooled DMEM media (Gibco)containing antibiotics and then removed into the hood. After washed with D-hank's solution three times, muscle biopsies were removed of fascia,tendon, nerve and blood vessels and minced into small pieces about 1-3 mm3,and then transferred into a centrifugation tube. 0.2% collgenase-type Ⅺ(Sigma) and 0.1% pancreatin were added to the left tissue . Skeletal muscle cells of the rats were isolated with collagenase. ② Differential attachment was used to purify the skeletal muscle cells of the rats. After screened with cell screen cloth , cell suspension was transferred into a polylysine-coated flask and cultured at 37 ℃ in a humid atmosphere with 0.05 CO2 in air for 1 hour. All the cells that did not adhere to the flask were then transferred to another flask with new culture medium and cultured for approximately 1 hour at 37 ℃ .Again,the non-adhering cells were transferred to another flask and were incubated at 37 ℃ overnight.The technique was carried out for an additional 4-5 days. This operation was repeated every 24 hours until the fifth day. Those cells attached on days 5-6 were MDSCs.③ After they grew with 70% confluence , the cells were digested with trypsin and passaged at the ratio of 1:2. After digestion, the generative cells were inoculated to the culture plate with 6-well cover glass. Growing culture medium was added and cell slide was prepaared 24 hours later. The expression of specific protein antigen and stem cell antigen-1 of the cultured cells were identified with immunohistochemical staining.MAIN OUTCOME MEASURES: Morphology and identification of MDSCs.RESULTS: ① Morphology of MDSCs: Cells isolated from skeletal muscle tissue took the form of spheres and had high refraction. When subcultured,they began to adhere at the 12th hour, at that time they were still round,and complete their attachment at the 48th hour. Then they began to expand and ultimately became fusiform-shaped or spindle-shaped, having two poles and small size. As time going, they fused with each other to form mature poly-nuclear myotubes. ② Identification results of muscle-derived stem cells: MDSCs were desmin and stem cell antigen-1 (Sca-1) positive specific for some stem cells. Red fluorescence effluenced from cytoplasm was found under the fluorescence microscope and the positive rate reached 90%.CONCLUSION: MDSC belongs to adult stem cells and has the advantages of rich source, low immunogenicity and long survival after transplantation. High-purity muscle-derived stem cells can be obtained through primary culture , and immunohistochemical technique can identify muscle-derived and specific characteristics of stem cells, so it has a broad application perspective in tissue engineering and gene therapy.

Extracellular matrix for the replacement of ureteral defect / 生物医学工程学杂志

This is an experiment on rabbits to evaluate the possibility of ureteral replacement by extracellular matrix. We adopted a biochemical method for preparing a new tissue engineering material named Extracellular Matrix (ECM), and the ECMs were used as homologous grafts to replace the defect in the ureters. Light microscopy, scanning electron microscopy, immunohistochemical technique and intravenous urography were used. The routine blood and biochemical laboratory tests were made before and after operation, and the measured values of pressure in the ureter of experiment and control groups were compared. The ureteral ECM was found in the experiment to promote the regeneration of all ureteral wall components. There were no significant differentces between the regenerative tissue and the normal tissue in morphology and function 16 weeks after replacement. The homologous ECM might be an ideal replacement material for ureteral defect.

Transplantation of adrenocortical cells in allorat using porous PHB as cell carrier / 生物医学工程学杂志

This experiment was designed to investigate the feasibility of transplanatation of using porous PHB as cell carrier for the transplanatation of adrenocortical cells. Adrenocortical cells from rat adrenal gland were separated and cultured in vitro. The effect of PHB on the proliferation and secretory function of adrenocortical cells were evaluated by MTT and RIA methods. Then adrenocortical cells were seeded into porous PHB. After the cells were cultured in vitro for about seven days, they were implanted into the rats having undergone bilateral adrenalectomy. The changes of blood corticosterone and aldosterone and the local histological changes in these rats were observed. Adrenocortical cells were able to grow and survive on PHB. No effect on the proliferation and secretory function of adrenocortical cells were observed. Most bilateral adrenalectomized rats bearing the transplanted adrenocortical cells within PHB (study group) survived longer than did the adrenalectomized rats in control group. The blood corticosterone level and aldosterone level of study group were higher than those of control group. It was found that PHB has no effects on the survival, proliferation and secretory function of adrenocortical cells. Adrenocortical cells within PHB can survive a period of time and can secrete corticosterone and aldosterone which can meet the needs of the adrenalectomized rats. PHB can degrade slowly in vivo. It is feasible to perform transplantation of adrenocortical cells using porous PHB as cell carrier.