Exosomes Derived from Human Amniotic Mesenchymal Stem Cells Facilitate Diabetic Wound Healing by Angiogenesis and Enrich Multiple lncRNAs

BACKGROUND@#Diabetic wound healing remains a major challenge due to the impaired functionality of angiogenesis by persistent hyperglycemia. Mesenchymal stem cell exosomes are appropriate candidates for regulating the formation of angiogenesis in tissue repair and regeneration. Here, we explored the effects of exosomes derived from human amniotic mesenchymal stem cell (hAMSC-Exos) on the biological activities of human umbilical vein endothelial cells (HUVECs) treated with high glucose and on diabetic wound healing and investigate lncRNAs related to angiogenesis in hAMSC-Exos. @*METHODS@#hAMSCs and hAMSC-Exos were isolated and identified by flow cytometry or western blot. A series of functional assays such as cell counting kit-8, scratching, transwell and tube formation assays were performed to evaluate the potential effect of hAMSC-Exos on high glucose-treated HUVECs. The effect of hAMSC-Exos on diabetic wound healing were tested by measuring wound closure rates and immunohistochemical staining of CD31. Subsequently, the lncRNAs profiles in hAMSC-Exos and hAMSCs were examined to screen the lncRNAs related to angiogenesis. @*RESULTS@#The isolated hAMSC-Exos had a size range of 30–150 nm and were positive for CD9, CD63 and CD81. The hAMSC-Exos facilitate the functional properties of high glucose-treated HUVECs including the proliferation, migration and the angiogenic activities as well as wound closure and angiogenesis in diabetic wound. hAMSC-Exos were enriched lncRNAs that related to angiogenesis, including PANTR1, H19, OIP5-AS1 and NR2F1-AS1. @*CONCLUSION@#Our findings demonstrated hAMSC-Exos facilitate diabetic wound healing by angiogenesis and contain several exosomal lncRNAs related to angiogenesis, which may represent a promising strategy for diabetic wound healing.

Exosomal microRNAs from Mesenchymal Stem Cells: Novel Therapeutic Effect in Wound Healing

BACKGROUND@#Wound healing is a complicated biological process that leads to the regeneration of damaged skin tissue. Determining the methods to promote wound healing has become a hot topic in medical cosmetology and tissue repair research. Mesenchymal stem cells (MSCs) are a group of stem cells with the potential of self-renewal and multidifferentiation. MSCs transplantation has a broad application prospect in wound healing therapy. Many studies have demonstrated that the therapeutic capacity of MSCs is mainly mediated by paracrine actions. Exosomes (EXOs), which are nanosized vesicles carrying a variety of nucleic acids, proteins and lipids, are an important component of paracrine secretion. It has been demonstrated that exosomal microRNAs (EXO-miRNAs) play a key role in the function of exosomes. @*METHODS@#In this review, we focus on current research on miRNAs from MSC-derived exosomes (MSC-EXO miRNAs) in terms of sorting, releasing and function and their effects on inflammation regulation, epidermal cell function, fibroblast function, and extracellular matrix formation. At last, we discuss the current attempts to improve the treatment of MSC-EXO-miRNAs. @*RESULTS@#Many studies have demonstrated that MSC-EXO miRNAs play a key role in promoting wound healing. They have been shown to regulate inflammation response, enhance epidermal cell proliferation and migration, stimulate fibroblast proliferation and collagen synthesis, and regulate extracellular matrix formation. Besides, there have been a number of strategies developed to promote MSC-EXO and MSC-EXO miRNAs for wound healing treatment. @*CONCLUSION@#Utilizing the association of exosomes from MSCs with miRNAs may be a promising strategy to promote trauma healing. MSC-EXO miRNAs may provide a new approach to promote wound healing and improve the quality of life for patients with skin injuries.

Extracellular Vesicles from Adipose-Derived Stem Cells Promote Diabetic Wound Healing via the PI3K-AKT-mTOR-HIF-1a Signaling Pathway

BACKGROUND@#Impaired potential of hypoxia-mediated angiogenesis lead poor healing of diabetic wounds. Previous studies have shown that extracellular vesicles from adipose derived stem cells (ADSC-EVs) accelerate wound healing with unelucidated mechanism. However, it is not yet clear about the underlying mechanism of ADSC-EVs in regulating the hypoxia-related PI3K/AKT/mTOR signaling pathway of vascular endothelial cells in diabetic wounds. Therefore, in this study, human derived ADSC-EVs (hADSC-EVs) isolated from adipose tissue were co-cultured with advanced glycosylation end product (AGE) treated human umbilical vein endothelial cells (HUVECs) in vitro and local injected into the wounds of diabetic rats. @*METHODS@#In vitro, the therapeutic potential of hADSC-EVs on AGE-treated HUVECs was evaluated by cell counting kit-8, scratching, and tube formation assay. Subsequently, the effects of hADSC-EVs on the PI3K/AKT/mTOR/HIF-1a signaling pathway were also assayed by qRT-PCR and western blot. In vivo, the effect of hADSC-EVs on diabetic wound healing in rats were also assayed by closure kinetics, Masson staining and HIF-1α-CD31 immunofluorescence. @*RESULTS@#hADSC-EVs were spherical in shape with an average particle size of 198.1 ± 91.5 nm, and were positive for CD63, CD9 and TSG101. hADSC-EVs promoted the expression of PI3K-AKT-mTOR-HIF-1a signaling pathway of AGEs treated HUVECs with improved the potential of proliferation, migration and tube formation, and improve the healing and angiogenesis of diabetic wound in rats. However, the effect of hADSC-EVs described above can be blocked by PI3K-AKT inhibitor both in vitro and vivo. @*CONCLUSION@#Our findings indicated that hADSC-EVs accolated the healing of diabetic wounds by promoting HIF-1α-mediated angiogenesis in the PI3K-AKT-mTOR depend manner.

Knockdown the expression of ku70 and lig4 in HEK293T cells by CRISPR/Cas13 system / 生物工程学报

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.

Advances in the research of effects of competing endogenous RNAs and their regulatory networks in pathological scars of skin / 中华烧伤杂志

The skin pathologic scar is a skin fibrous proliferative disease characterized by abnormal proliferation of fibroblasts and overdeposition of extracellular matrix. Unclarity of genesis and development mechanism is the main reason that restricts its diagnosis and treatment. In recent years, it has been found that microRNAs play important roles in the regulation mechanism of pathological scars. The competing endogenous RNAs (ceRNAs) have microRNA response elements which can be competitively combined with microRNAs through sponge adsorption. Through the mutual regulation of RNAs, ceRNAs regulate the expression of target gene and participate in the development of disease. Based on the ceRNA hypothesis, this paper systematically reviews the biological functions and clinical significance of ceRNAs in pathological scars of skin, and discusses the role of ceRNAs and " RNA-microRNA-RNA" regulation network in pathologic scars. The ceRNA therapy may become a new model therapy for skin scars in the future.

Advances in the research of the application of induced pluripotent stem cells in tissue engineering skin as seed cells / 中华烧伤杂志

Tissue engineering skin has a wide application prospect on the clinical treatment of all sorts of skin defect, especially large area burn. The shortage of seed cells and their function improvement are the main problems in this field. The existing seed cells of tissue engineering skin are difficult to meet the needs of clinical application due to the limitations of acquisition, proliferation, and aging. Subsequently, the generation of induced pluripotent stem cells (iPSCs) provides a safe and efficient cell source for tissue engineering skin. Our article focuses on the origin of iPSCs and its characteristics of differentiating into keratinocytes, fibroblasts, melanocytes, vascular smooth muscle cells, nerve cells, and hair follicle, and discusses the main problems and prospects of iPSCs in establishment of tissue engineering skin and application in wound repair.

Advances in the research of effects of exosomes derived from stem cells on wound repair / 中华烧伤杂志

Exosomes are nano-vesicles released by many kinds of cells. Exosomes play a significant role in cell-to-cell communication and substance transportation through direct effect of signaling molecules on the cell membrane surface, intracellular regulation of cellular content during membrane fusion, or regulation of release of various bioactive molecules. Several studies have reported that culture supernatant of stem cells has some related exosomes to take part in wound repair. The secretion of exosomes is depended on the source and the physiological and pathological condition of deriving cells. How to stimulate the stem cells to produce exosomes maximally and their clinical application are worthy to explore. In this review, we summarize the biological function and application of exosomes derived from stem cells in wound repair.

Advances in the research of effects of regulation of cell autophagy on wound healing / 中华烧伤杂志

As one of the self-protection mechanism, autophagy widely exists in eukaryotic cells. It plays an important role in maintaining cells survival, update, material recycling, and tissue homeostasis. A series of researches discovered that autophagy played dual function in fibrotic diseases. The induction of autophagy can promote the degradation of collagen on one hand, on the other hand, the regulation of autophagy through microRNA, transforming growth factor β, and other factors can promote the occurrence of fibrosis. In wound healing, autophagy may participate in the pathophysiological processes of inflammation, reepithlialization, and wound remodeling. The regulation of cell autophagy may become an effective way and the new target for treatment of wound and pathological scar.

Screening and bioinformatics analysis of differentially expressed genes in hyperplastic scar / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen differentially expressed genes in hyperplastic scar to explore the pathogenesis of hyperplastic scar and identify new therapeutic targets.</p><p><b>METHODS</b>Three pairs of surgical specimens of hyperplastic scar and adjacent normal skin tissues were collected to investigate the differentially expressed genes in hyperplastic scar using Agilent gene oligonucletide microarray and clustering analysis. DAVID Bioinformatics Resources 6.7 was used for GO analysis and pathway analysis.</p><p><b>RESULTS AND CONCLUSION</b>Distinctly different gene expression profiles were found between hyperplastic scar tissues and normal skin tissues. Compared with normal skin tissue, hyperplastic scar tissues showed 3142 up-regulated and 2984 down-regulated genes by two folds and 28 up-regulated and 44 down-regulated genes by 5 folds after repeating the experiment once; after repeating the experiment twice, 3004 genes were found up-regulated and 3038 down-regulated by 2 folds and 25 up-regulated and 38 down-regulated by 5 folds in hyperplastic scars. In all the 3 specimens, 1920 genes were up-regulated and 1912 down-regulated by 2 folds and 18 up-regulated and 29 down-regulated by 5 folds. The dysregulated genes in hyperplastic scar were involved in cell cycles, cell proliferation, immune response and cell adhesion (CDKN1C, CDKN2A, CTNNA3, COL6A3, and HOXB4) and in signaling pathway of focal adhesion, TGF-beta signaling pathway, p53 signaling pathway, cell cycle, and tumor-associated pathways (TGFβ1, CDKN1C, CDKN2A, CDC14A , ITGB6, and EGF).</p>

Expression of microRNA-203 and P63 in human epidermal stem cells and keratinocytes / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the changes in expression of microRNA-203 and P63 in human epidermal stem cells and KCs, and to investigate their effects and significance in the epidermal proliferation and differentiation.</p><p><b>METHODS</b>(1) Five normal foreskin tissue specimens were collected from 5 patients by circumcision in Department of Urinary Surgery of the First Affiliated Hospital of Nanchang University from March to June in 2013. Then single cell suspension was obtained by separating epidermis with trypsin digestion method. The cells were divided into quick adherent cells and non-quick adherent cells by type IV collagen differential adherent method. The biological characteristics of cells were observed by inverted phase contrast microscope immediately after isolation and on post culture day (PCD) 3. The expression of CD29, keratin 19, keratin 1, and keratin 10 was identified by immunocytochemical staining. The expression of microRNA-203 and mRNA of P63 was determined by real-time fluorescent quantitative RT-PCR. The protein expression of P63 was determined by Western blotting. Data were processed with t test and Pearson correlation analysis.</p><p><b>RESULTS</b>(1) Immediately after isolation, quick adherent cells were small, round, and dispersed uniformly. On PCD 3, the cells adhered firmly, and they grew in clones. Immediately after isolation, non-quick adherent cells appeared in different shapes and sizes, and dispersed unevenly. On PCD 3, the cells adhered precariously and did not show clonal growth. Quick adherent cells showed positive expression of CD29 and keratin 19, while non-quick adherent cells showed positive expression of keratin 1 and keratin 10. Quick adherent cells were identified as epidermal stem cells, and non-quick adherent cells were identified as KCs. (2)The expression level of microRNA-203 in epidermal stem cells (0.74 ± 0.20) was lower than that in KCs (3.66 ± 0.34, t =16.582, P <0.001). The mRNA expression level of P63 in epidermal stem cells (4. 16 ± 0.28) was higher than that in KCs (2.90 ± 0.39, t =5. 850, P =0.001). The protein expression level of P63 in epidermal stem cells (1.42 ± 0.05) was higher than that in KCs (0.73 ± 0.03, t =26.460, P <0. 001). (3) The expression level of microRNA-203 was in significantly negative correlation with the expression levels of mRNA and protein of P63 (with r values respectively - 0. 94 and -0.98 , P values below 0.05).</p><p><b>CONCLUSIONS</b>The expression levels of microRNA-203 and P63 in human epidermal stem cells and KCs were significantly different, which might be related to the different characteristics of proliferation and differentiation of the cells.</p>

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on wound healing and microRNA expression in diabetic rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) on wound healing and microRNA expression in diabetic rats.</p><p><b>METHODS</b>Eighteen male SD rats of clean grade were used to reproduce diabetes model. Four weeks later, a total of 64 full-thickness skin wounds were created on the back of 16 rats with established diabetes, with 4 wounds on each rat. Two symmetrical wounds on either side of the spine were created as a pair according to paired design. Then the wounds were divided into groups A and B according to the random number table and blind method (red and blue tags on the rhGM-CSF or the gel vehicle), with 32 wounds in each group. The ointment with red tag was applied on the wounds of group A and the blue one on group B. The application was conducted once a day, with a thickness of 3 mm, up to post injury day (PID) 14. Gross observation of wound healing was conducted on PID 3, 7, 14. The wound healing rate was determined on PID 3 and 7. On PID 3, 7, 14, tissues from 2, 4, and 8 wounds were harvested from each group respectively for the observation of the histopathological changes with HE staining, and also for analyzing the expression of proliferating cell nuclear antigen (PCNA) and CD31 with immunohistochemical staining (denoted as absorbance value). On PID 7, tissues from 6 wounds in each group were harvested for microarray gene chip to screen the differentially expressed microRNAs. Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway on the differentially expressed microRNAs were performed after the microRNA screening results were validated by real-time fluorescent quantitative RT-PCR. Data were processed with paired t test or two-sample t test.</p><p><b>RESULTS</b>(1) On PID 3, the wound area was significantly decreased, and the wound granulation was significantly proliferated in both groups. On PID 7, the wound area was further decreased, and the wound area was almost filled by granulation in both groups; the conditions in group A were better. On PID 14, all the wounds in group A were almost healed, while a small area of raw wound with incrustation still remained in some wounds of group B. On PID 3 and 7, the wound healing rates of group A were (41 ± 5)% and (75 ± 4)%, significantly higher than those of group B [(31 ± 9)% and (71 ± 4)%, with t values respectively 10.13 and 8.06, P values below 0.001]. (2) On PID 3, the epidermal cells, endothelial cells, and Fbs in the wounds of 2 groups were sparse, with heavy infiltration of inflammatory cells. The above condition in the wounds was better in group A than in group B. On PID 7, the epidermal cells, endothelial cells, and Fbs were gradually well arranged in group A; infiltration of inflammatory cells decreased, and the condition was better than that of group B. On PID 14, the wounds of group A were completely covered by epidermis, while infiltration of inflammatory cells still remained in some wounds of group B. (3) On PID 3, 7, 14, the positive expressions of CD31 and PCNA in group A were respectively 0.275 ± 0.018, 0.345 ± 0.034, 0.305 ± 0.023; 0.406 ± 0.063, 0.223 ± 0.011, 0.045 ± 0.022. They were significantly higher than those of group B (0.222 ± 0.020, 0.229 ± 0.018, 0.197 ± 0.015; 0.324 ± 0.039, 0.162 ± 0.012, 0.018 ± 0.020, with t values from 2.281 to 9.652, P < 0.05 or P < 0.01). (4) According to the microRNAs detection and screening, as compared with group B, 18 microRNAs were up-regulated while 13 were down-regulated in the wounds of group A. (5) The results of real-time fluorescent quantitative RT-PCR had good consistency with the results of microRNAs detection. (6) Enrichment analysis of KEGG signaling pathway showed that among the 31 differentially expressed microRNAs, 4 took part in the MAPK signaling pathway, 3 took part in the Wnt signaling pathway, 1 took part in the TGF-β signaling pathway, 3 took part in the epidermal growth factor receptor signaling pathway, 2 took part in the cell cycle pathway, 5 took part in the axon guidance signaling pathway, 6 took part in the focal adhesion pathway, 3 took part in the regulation of actin cytoskeleton pathway, 1 took part in the extracellular cell matrix receptor pathway, 3 took part in the adherens junction pathway, and 1 took part in the cell adhesion molecules pathway. After disclosing the blind, it showed that the ointment with red tag was the rhGM-CSF gel and the blue one was gel vehicle.</p><p><b>CONCLUSIONS</b>The rhGM-CSF gel can promote wound healing in diabetic rats, producing significant differential microRNA expression in wounds, and they may be the target at gene post-transcriptional level of rhGM-CSF gel in promoting wound healing.</p>

Different expression profiles of micro-RNA in epidermal cells of different developmental stages / 中华创伤杂志

Objective To investigate the difference in expression profiles of micro-RNA (miR-NA) between human epidermal stem cells and epidermal keratinocytes.Methods (1) Human primary epidermal stem cells and keratinocytes were obtained with enzyme digestion method and type Ⅳ collagen coated chosen method.Growth of cells cultured in vitro was observed by inverted microscope.Monoclonal antibody of integrinβ1,keratin 1 (CK1),CK10,and CK19 were detected by immunocytochemical staining.(2)Total RNA was respectively isolated from epidermal stem cells and epidermal keratinocytes by Trizol-based single-step procedure,detected by formaldehyde denaturing gel electrophoresis,purified by mirVanaTM miRNA isolation kit,and then labeled and hybridized by miRNA labeling and hybridization kit.Images of hybridization were analyzed using the Feature Extraction (Version 10.7).Data normalization and difference analysis were performed with GeneSpring (GX 10.0).Moreover,miRNA microarray results were confirmed by RT-PCR.(3) Target genes of differently expressed miRNA were predicted.Results Epidermal stem cells exhibited rapid adherence to type Ⅳ collagen and formed distinct clones after 3 days of culture; expressions of integrinβ1 and CK19 were positive.Keratinocytes could not adhere rapidly to type Ⅳ collagen and formed few clones after 3 days of culture ; expressions of CK1 and CK10 were positive.(2) Of the epidermal stem cells,31 miRNAs were up-regulated and 153 down-regulated.Besides,significantly up-regulated miRNAs were hsa-miRNA-125b-3p,hsa-miRNA-197-5p,and hsa-miRNA-376a-3p,while significantly down-regulated miRNAs were hsa-miRNA-203,hsa-miRNA-29b-3p,and hsa-miRNA-34a-3p.Findings of RT-PCR on hsa-miRNA-197-5p and hsa-miR-29b-3p revealed high concordance with the microarray results.(3) Some miRNAs target genes indicated that miRNA related to cell proliferation,differentiation,apoptosis,aging and other biological characteristics.Conclusion Distinct differences in miRNA expression profiles are detected between human epidermal stem cells and keratinocytes and this may correlate closely with their different biological characteristics such as proliferation and differentiation ability.

Proliferation changes of human epidermal stem cells in response to transfection with human telomerase reverse transcriptase gene / 中华创伤杂志

Objective To investigate the changes of human epidermal stem cells after transfected with human telomerase reverse transcriptase(hTERT)gene.Methods The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT were transfected into in vitro cultured human fetal epidermal stem cells by liposome-mediated transfection.Then,the positive cells were selected with G418.The mRNA and protein expressions of hTERT were detected by reserve transcriptase-polymerase chain reaction(RT-PCR)and Western blot.The telomerase activity and the proliferation and cycle of human epidermal stem cells were detected by telomeric repeat amplification protocol(TRAP)-ELISA and flow cytometry respectively.Results RT-PCR and Western blot techniques detected weak mRNA and protein expressions of hTERT gene in untransfected and vacant vector transfected cells but high level of mRNA and protein expressions of hTERT gene in pIRES2-EGFP-hTERT transfected cells.Compared with untransfected and vacant vector transfected cells,the pIRES2-EGFP-hTERT transfected cells had higher telomerase activity,with lower proportion of cells at G_0/G_1 phase,higher proportion of cells at S and G_2/M phases and enhanced proliferation ability.Conclusion Transfection with hTERT gene can markedly enhance mRNA and protein expressions,telomerase activity and proliferation ability of hTERT gene of human epidermal stem cells euhured in vitro.

Effective research about local oxygen therapy to promoting renovation of residual surface of burns wound / 中国实用护理杂志

Objective In order to know the effective of local oxygen therapy to promoting renovation of residual surface of bums wound.Methods Divided 60 patients with burns into the experimental group and the control group,there were 30 cases in the each group.Routine nursing cares were used in the control group,while the local oxygen therapy was used in the experimental group.Compare the condition of secretion of wound between the two groups.Results The wound healing time of experimental grou was(5.87±3.29)d,which significant shorten than that of in the control group,the kinds of bacteria in the control group was more than in the experimental group.Conclusions The local oxygen therapy can effective promote the healing of residual surface of bums wound,which can reduce the infection rate of bacteria.

Structure and properties of tissue-engineered skin / 中国组织工程研究

In the human body, the structure of the organ and tissue is its functional foundation. With the human body skin losing, the skin also loses its function. When the skin defect area is quite large, it is the question that the source of the autologous skin is deficient. But with the engineering research progressing, the application of artificial skin to repair the skin defect is possible. In order to make the tissue-engineered skin have "normal" skin function, the structure research also should make them approach as far as possible to be similar. This article reviews the constructs, properties and applications of the epidermal substitutes, the dermal substitutes and the skin substitutes. With continued development of multi-discipline and continued progress of basic and clinical research, the constituted tissue-engineered skin substitutes will more approach the normal skin in the structure and properties.

Separation and culture of rabbit vaginal smooth muscle cells in vitro / 中国组织工程研究

BACKGROUND:Vaginal smooth muscle cells are mainly cultured by enzyme digestion method and explant method.The former method requires a short culture time with a high output,but this method demands a large number of tissues with a high opportunity of pollution,and the optimal digestion time is difficult to control.The latter method is simple and effective,but the culture needs a long time.OBJECTIVE:To observe the culture time of rabbit vaginal smooth muscle cells using explant + enzyme digestion methods,and to compare with the explant method.DESIGN,TIME AND SETTING:The in vitro observation experiment was performed at the Burn Institute and Animal Laboratory of First Affiliated Hospital,Nanchang University from February 2005 to February 2006.MATERIALS:Female New Zealand rabbits aged 4-5 months,with the body mass of 2.0-2.5 kg were selected for this study.METHODS:Vaginal smooth muscle cells using explant method:1 mm?1 mm?1 mm explants were uniformly incubated in a culture medium,and then placed in a incubator at 37 ℃ for 3.0-4.0 hours.After tissue confluence,tissues were immersed in DMEM containing 0.1 volume fraction,stored at 37 ℃ for 3.0-4.0 days.Following cells grew from the tissue islets,the medium was changed about once every three days.Vaginal smooth muscle cells using explant + enzyme digestion methods:The tissue pieces were digested by 0.1% collagenase type Ⅳ at 37 ℃ for 0.5-1 hour,and then enzymatic digestion was interrupted when the edge of tissue pieces became coarse.These pieces were cultivated in culture dishes.The remaining steps were the same as the explant method.Serial subculture:The 1st subculture was made when 50%-60% cells were confluent.After the 2nd passage of cells was obtained,subculture was made when 80% cells were confluent.MAIN OUTCOME MEASURES:Time of primary culture;Surface structure and ultrastructure of the 3rd passage of vaginal smooth muscle cells.RESULTS:The eruption time and the confluent time of vaginal smooth muscle cells could be shortened by the explant + collagen digestion methods about 1-2 days and 3-4 days respectively compared with the explant method only.Vaginal smooth muscle cells,which were obtained by the two methods mentioned above,could propagate for 5-6 passages.The attached cells were polygonal or spindle and after confluence could be seen,the marked smooth muscle cells "Peak-Valley" structure in some domains under the inverted microscope.The third passage of smooth muscle cell nuclei were serration;the cytoplasm contained the marked smooth muscle cell structural myofilaments and dense bodies and plenty of golgi bodies;the rough endoplasmic reticulum broadened;the free ribosome were abundant under the transmission electron microscope.This indicated that vaginal smooth muscle cells with synthesis phenotype could be greatly collected.CONCLUSION:Explant + enzyme digestion methods need a short cycle of primary culture.Vaginal explants should be kept moisture during drawing materials,isolation and the whole.The time of enzymatic digestion should not be too long,to the point that palpi pilosi is seen surrounding the explants.Primary culture should remain static state and use of plastic culture dishes.