| Objective:(1)The epidermal stem cells has been isolated, cultured and identified.(2)Exploring which can we inducing mature skin cells to the epidermal stem cells by improve the expression of cell cycle protein D1, at the same time,we should observe the change of cell morphology, phenotype, proliferation.(3)Exploring the epidermal cells which induced by the CCND1 can improve the skin wound of the nude mice.Method:(1)We has taken some fresh adult foreskin tissues of parents whose foreskin is too long. The tissues are digested Respectively by the decomposition enzyme(Dispase?)and trypsin, and they are filtered by 200 mesh. Then the cells is suspended by the Epilife complete medium and cultivated in the bottle that packed by the collagen type?. 30 minutes later in liquid, we wash away the floating cells, rejoin the Epilife medium and cultivate in the incubator. We detect situation about the expression of CK19, beta1 and CK10 by immunofluorescence technique.( 2) With the plasmid PEGFP-N1 and cell cycle protein D1 gene we synthesize expression plasmid PEGFP-N1-CCND1, then transfecte mature skin cells by the Lipofectamine ? 2000 and at last induce CCND1 gene of mature skin cells expression. Setting epidermal cells after transfection by PEGFP-N1-CCND1 as experimental group, epidermal stem cells as the positive control group, and epidermal cells by empty carrier PEGFP-N1 transfection as the negative control group. Record each group cells changes after transfection by cell count plate 5 days later. Testing the situation about each cell marker of CK19, beta 1 and CK10 on the expression of m RNA levels By RT-PCR. Detecting the situation about each cell marker of CK19,beta 1 and CK10 on the protein expression levels by Western Blot technique.Detecting the changes of protein expression about each group of epidermal stem cell marker antigen CK19, beta 1 and mature skin cell marker antigen CK10 by the cellimmunofluorescence techniques.(3)Divided 30 nude mices into 3 groups randomly, and setting experimental group,positive control group and the negative control group according to each cell by corresponding transplantation. We anesthetize the nude mice with 2% chloral hydrate by intraperitoneal injection and make a full thickness cutting injury of diameter 6 mm.We dyeing the cells of the experimental group, the positive control group and the negative control group with hoescht33342, then we blending the 5 x 106 cells and0.05 ml substrate glue and implant them to the wound of the nude mices. At last we observe the wound healing of the nude mices 10 days and 20 days later.At the same time, we cut the nude mices wound and observe the location of the epidermis cells in the nude mices wound by the frozen section of immunofluorescence technique.Result:(1)Epidermal stem cell suspension inoculation in the cultivation of the collagen type ? cover bottle after 30 min, and some cells grow small stick wall in training at the bottom of the bottle. As epidermal stem cells has a stronger affinity with collagen type ?, the epidermal stem cells is the first one which stick the bottom of the bottle,then we have washed the epidermal cells that not stick the wall by the PBS,at last we cultivate the epidermal cells in the constant temperature incubator. Epidermal stem cells has a small body, a big nucleus, a crumb grow, a rapid proliferation and a cell larger density, when the cells has paved the bottle, the cell interval become smaller and smaller. Cell immunofluorescence test results showed that epidermal stem cell marker protein CK19, beta 1 fluorescent has a positive expression, but the mature skin cells fluorescent marker protein CK10 expresses negatively.(2) When the epidermal stem cell has been cultured 5 to 6 generations, it has a bigger body, smaller nuclei, a decreasing nucleoplasm ratioand the cell proliferation is slow or even stagnation. The epidermal cells which has been transfection by the PEGFP-N1-CCND1 has a smaller body, a bigger nuclei, an increasing ratio and a fast proliferation. Cell immunofluorescence test results showed that the experimental group of cells and positive control group of epidermal stem cell marker protein CK19,beta 1 fluorescent expresses positively, and mature skin cells fluorescent marker protein CK10 expresses negatively; Epidermal stem cell marker protein CK19 expresses negatively, beta 1 fluorescent expresses negatively, mature skin fluorescent marker protein CK10 expresses positively. On the m RNA and protein levels RT-PCR and Western Blot technology tests reveals: CK19 and beta 1 express positively on the positive control group and experimental group cells, but the CK10 expresses negatively; the CK19 and beta 1 express negatively, but the CK10 expresses positively. The number of the experimental group and positive control group cells are significantly greater than the negative control group 5 days later.( 3) After continuous observation about the wound healing after nude mouse transplantation we can see : The wound area of the experimental group and the positive control group in nude mice after 10 d is smaller than the negative control group; The wound area of the experimental group and the positive control group after20 d nude mice is smaller than the negative control group. Frozen section immunofluorescence showed that CK19 and beta 1 of the experimental group and the positive control group in nude mice wound express positively, CK10 expresses negatively; CK19 and beta 1 of negative control group in nude mice wound to expresses negatively, CK10 expresses weakly.Conclusion:(1) Taking advantage of the epidermal stem cells and other cells of epidermis' s difference of collagen type ? affinity,we can obtain the high purity epidermal stem cells.(2)The method that putting PEGFP-N1-CCND1 into mature skin cells can improve expression of mature skin cells' s CCND1 by liposome can induction of mature skin cells to differentiation of epidermal stem cells.(3)The epidermal cells which has been induced by the PEGFP-N1-CCND1 have similar ability that can promote the nude mouse wound healing. |
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