OBJECTIVETo explore the biological characteristics of fibroblasts in adult granulation tissue in vitro, including cell viability, CD phenotypic, factor and protein expression, and differentiation, so as to facilitate further research of the role of fibroblasts in early wound healing.

METHODSFibroblasts were isolated from human granulation tissue and cultured by mechanical and enzymatic digestion method. The cell morphology and proliferation were observed under inverted phase contrast microscope. The first and third passages of cells' growth curve were drawn respectively. The surface markers (CD105, CD73, CD90, CD44, CD34, CD45, CD19, CD11b, HLA-DR) of the first and third passage fibroblasts were identified by Flow Cytometry. And the expression of Vimentin, CK19, CD31 and Factor VIII were detected by immunocytochemistry.

RESULTSPrimary cultured fibroblasts were short spindle, polygonal and irregular in shape. The morphology of fibroblasts were uniform by repeatedly passage cultured in vitro and showed spindle-shaped. The proliferative capacity of the fibroblasts were not significantly different, with logarithmic growth phase. From 1 to 4 days, The primary and third passage cells' proliferation was no difference (P > 0.05), After the five days, the proliferative ability of third generation was better than the primary passage (P < 0.01). All fibroblasts highly expressed mesenchymal stem cells' surface markers CD105, CD73, CD90 and CD44, and didnt express hematopoietic stem cells' surface markers CD34, CD45, CD19, CD11b and HLA-DR. The expression of mesenchymal stem cells' surface markers in third generation of cells were increased significantly. Immunocytochemistry showed positive expression of Vimentin, CD31, and negative expression of CK19 and Factor VIII.

CONCLUSIONSFibroblasts in human granulation tissue show the biology characteristics of Mesenchymal Stem Cells. Some biological markers of endothelial cells are expressed in fibroblasts in granulation tissue. The fibroblasts may play an important role during the process of endothelial to mesenchymal transformation in early wound healing.

Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; Gene Expression ; Humans

Objective To explore the correlated factors and clinical features of cognitive impariment in parkinson's disease (PD).Methods A total of 419 patients with PD were collected from Xiangya Hospital of Centre-South University during Mar 1st,2017 to Nov 30th,2017.The cognitive functions of patients were assessed with the Mini-Mental State Examination (MMSE),and the basic information and the motor symptoms of 419 PD patients were selected at the same time.The PD patients were classified into three groups according to the MMSE score:PD with no cognitive impairment (PD-NC),mild cognitive impairment in PD (PD-MCI),and Dementia in PD (PD-D).The data were analyzed by SPSS 20.0.Results There were 156 patients with PD-MCI (37.2％) and 64 patients with PD-D (15.3％).The difference of sex and disease duration among three groups were not statistically significant (P ＞ 0.05).The significant difference was found among PD-D,PD-MCI,and PD-NC groups in age of onset,age,educational attainment,Unified Parkinson's disease Rating Scale (UPDRS)-Ⅱ score,UPDRS-Ⅲ score and Hoehn-Yahr stage (P ＜ 0.05).There were significant differences among three groups in MMSE score and its items (P ＜ 0.01).Logistics regression analysis found that the age of onset,educational attainment,and Hoehn-Yahr stage were the risk factors of cognitive impairment in PD patients (P ＜ 0.05).Conclusions Cognitive impairment is common in PD patients,and it is relevant to the age of onset,educational attainment and the severity of illness of PD patients.

Objective To preliminarily explore the effects of human dermis derived mesenchymal stem cells (hDMSCs) on expressions of α-smooth muscle actin (α-SMA) and decorin (DCN) in hypertrophic scars fibroblasts (HSFB) at different periods,and to explore the feasibility of MSCs in prevention and treatment of HSFBs.Methods hDMSCs were cultured with mechanical method combined with enzyme digestion.The cells of the third generation which were well grown were taken,and flow cytometry (FCM) was used to detect CD molecules in hDMSCs.Immunocytochemistry was used to detect cytokeratin 19 (CK19) and vimentin and identify the separated cells.The cells were differentiated into lipoblasts,chondroblasts and osteoblasts.According to the formation course of hypertrophic scar,the scar specimens were divided into 6-month,l-year,and 2-year group with three cases in each group.HSFBs from different groups were co-cultured with well-adherent hDMSCs of the third generation in non-contact transwell co-culture system for 21 days.And HSFBs from the corresponding groups were cultured in normal six-well plate as the controls.Real-time fluorescent-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expressions of mRNA and proteins of α-SMA and DCN in HSFBs from different groups.Results hDMSCs highly expressed the surface markers including CD73,CD105,CD44 and CD90,etc.,but did not express hematopoietic stem cell surface markers including CD 14,CD34 and CD45.They positively expressed vimentin but not CK19.The cells can be differentiated into lipoblasts,chondroblasts and osteoblasts,which was in line with the minimum identification standards of mesenchymal stem cells.For HSFB cultured in normal six-well plates,α-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 198.20 ± 15.46/0.29 ± 0.070,175.24 ± 17.04/0.38 ± 0.110,and 125.73 ± 6.99/0.33 ±0.085,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 61.30 ± 9.79/0.015 ± 0.003,70.89 ± 11.29/0.020 ± 0.007,and 77.31 ± 4.80/0.023 ± 0.003,respectively.For HSFB co-cultured with 5 × 104 hDMSCs,oα-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 48.40 ± 6.42/ 0.100 ± 0.020,192.16 ± 11.37/0.110 ± 0.014,and 73.33 ± 6.29/0.110 ± 0.016,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 156.92 ± 14.91/0.049 ±0.015,154.42 ± 18.17/0.033 ± 0.008,and 140.82 ± 7.32/0.030 ± 0.004,respectively.Compared with the control group(Cultured in normal six-well plates),mRNA and protein expressions of α-SMA in HSFBs were decreased after co-culture with 5 × 104 hDMSCs,mRNA and protein expressions of DCN were increased.Furthermore,it suggested that hypertrophic scar changed significantly in the early formation stage namely in the 6-month group.Conclusions hDMSCs can down-regulate α-SMA mRNA and protein expressions of HSFBs and up-regulate mRNA and protein expressions of DCN in the in-vitro culture system.Those effects were particularly obvious on fibroblasts at the early formation of hyperplastic scar.Anti fibrosis role of hDMSCs is expected to be used in increasing the healing quality of the wound and in the prevention and treatment of pathological scars.

Objective To preliminarily explore the effects of human dermis derived mesenchymal stem cells (hDMSCs) on expressions of α-smooth muscle actin (α-SMA) and decorin (DCN) in hypertrophic scars fibroblasts (HSFB) at different periods,and to explore the feasibility of MSCs in prevention and treatment of HSFBs.Methods hDMSCs were cultured with mechanical method combined with enzyme digestion.The cells of the third generation which were well grown were taken,and flow cytometry (FCM) was used to detect CD molecules in hDMSCs.Immunocytochemistry was used to detect cytokeratin 19 (CK19) and vimentin and identify the separated cells.The cells were differentiated into lipoblasts,chondroblasts and osteoblasts.According to the formation course of hypertrophic scar,the scar specimens were divided into 6-month,l-year,and 2-year group with three cases in each group.HSFBs from different groups were co-cultured with well-adherent hDMSCs of the third generation in non-contact transwell co-culture system for 21 days.And HSFBs from the corresponding groups were cultured in normal six-well plate as the controls.Real-time fluorescent-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expressions of mRNA and proteins of α-SMA and DCN in HSFBs from different groups.Results hDMSCs highly expressed the surface markers including CD73,CD105,CD44 and CD90,etc.,but did not express hematopoietic stem cell surface markers including CD 14,CD34 and CD45.They positively expressed vimentin but not CK19.The cells can be differentiated into lipoblasts,chondroblasts and osteoblasts,which was in line with the minimum identification standards of mesenchymal stem cells.For HSFB cultured in normal six-well plates,α-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 198.20 ± 15.46/0.29 ± 0.070,175.24 ± 17.04/0.38 ± 0.110,and 125.73 ± 6.99/0.33 ±0.085,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 61.30 ± 9.79/0.015 ± 0.003,70.89 ± 11.29/0.020 ± 0.007,and 77.31 ± 4.80/0.023 ± 0.003,respectively.For HSFB co-cultured with 5 × 104 hDMSCs,oα-SMA mRNA and protein expressions of HSFB in the 6-month,1-year and 2-year groups were 48.40 ± 6.42/ 0.100 ± 0.020,192.16 ± 11.37/0.110 ± 0.014,and 73.33 ± 6.29/0.110 ± 0.016,respectively;while DCN mRNA and protein expressions of HSFB in the corresponding groups were 156.92 ± 14.91/0.049 ±0.015,154.42 ± 18.17/0.033 ± 0.008,and 140.82 ± 7.32/0.030 ± 0.004,respectively.Compared with the control group(Cultured in normal six-well plates),mRNA and protein expressions of α-SMA in HSFBs were decreased after co-culture with 5 × 104 hDMSCs,mRNA and protein expressions of DCN were increased.Furthermore,it suggested that hypertrophic scar changed significantly in the early formation stage namely in the 6-month group.Conclusions hDMSCs can down-regulate α-SMA mRNA and protein expressions of HSFBs and up-regulate mRNA and protein expressions of DCN in the in-vitro culture system.Those effects were particularly obvious on fibroblasts at the early formation of hyperplastic scar.Anti fibrosis role of hDMSCs is expected to be used in increasing the healing quality of the wound and in the prevention and treatment of pathological scars.