Objective To investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
Objective To investigate the feasibility of differentiating human umbilical cord blood stem cells into hepatocytes. Methods Thirty-six BALB/c nude mice were randomly divided into experimental group and control group(18 in each of the group), and experimental group was again randomly divided into group A， B and C (six in each of the group). The mice in experimental group and control group were exposed to 350 cGy radiation produced by 60Ｃｏ. After 3 h, karyocytes at different concentrations in the fresh human umbilical cord blood were injected into the mice in experimental group A, B, C via their tail veins, and the equal volume of normal sodium (NS) was also injected into control group via tail veins. After one month, carbon tetrachloride (CCl4) was injected into experimental group A, B and control group via abdominal cavity, and the equal volume of normal sodium was injected into experimental group C. After two months, immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the expressions of human cytokeratin-18 (CK18), cytokeratin-19 (CK19) and albumin (ALB) in liver tissues of all mice. Results The expressions of CK18, CK19 and ALB in injured liver tissues were all positive, and the expressions of experimental group B were higher than those of experimental group A （P＜0.05）, but the expressions of CK18, CK19 and ALB in the liver tissues of control group and experimental group C, whose were not injured with CCl4, were all negative. Conclusion Human umbilical cord blood-derived stem cells can differentiate into hepatocytes and express ALB under special microenvironment after liver injured by CCl4 , and the expression level of ALB maybe directly related to the number of human umbilical cord blood stem cells.
Objective To compare the myogenic differentiation abil ity in vitro of rabbit adipose-derived stem cells (ADCSs) from different sites so as to provide ideal seed cells for repair and reconstruction of urinary tract. Methods Adipose tissues were obtained from the nape of the neck, post peritoneum, and vicinity of epididymis of a 4-month-old male New Zealand rabbit and ADSCs were harvested through collagenase digestion. ADSCs were purified by differential attachment method. The protein marker CD44 of rabbit ADSCs was used to identify the stem cells by immunocytochemistry, then the5th generation of ADSCs were induced to differentiate into adipogenic, osteogenic, and myogenic cells. Multi- differentiation was confirmed by Oil red O staining, von Kossa staining, and RT-PCR. Myogenic differentiation abil ities of ADSCs from 3 different sites were compared between the control group (L-DMEM medium containing 10%FBS) and the experimental group (myogenic medium) by RT-PCR method. Results ADSCs could be easily isolated from adipose tissues of the nape of the neck, post peritoneum, and vicinity of epididymis. ADSCs displayed a typical cobblestone morphology. Brown particles could be seen in ADSCs by CD44 immunocytochemistry staining. Oil red O staining showed red fat drops in ADSCs after 14 days of adipogenic culture. Black matrix could be seen in ADSCs by von Kossa staining after 28 days of osteogenic culture. RT-PCR detection showed moderate α-actin expression in the control group and b α-actin expression in the experimental group after 42 days of myogenic culture. The growth rate of α-actin from the adipose tissue of post peritoneum (28.622% ± 4.879%) was significantly lower (P lt; 0.05) than those from the adipose tissues of the nape of the neck (35.471% ± 3.434%) and vicinity of epididymis (38.446% ± 4.852%). Conclusion The ADSCs from different sites show different myogenic differentiation abil ities in vitro. ADSCs from the adipose tissues of the nape of the neck and vicinity of epididymis can be used as ideal seed cells for tissue engineering of lower urinary tract.
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.
Objective To observe the efficacy of all trans retinoic acid (ATRA) combined with 5-Fu on treating subcutaneously implanted gastric carcinoma SGC 7901 in the nude mice. Methods Thirty-two nude mice bearing tumor were randomly divided into 4 groups: control group (group A), ATRA 1 000 μg/d orally taken group (group B), 5-Fu 27 mg/kg group peritoneally injected (group C), 5-Fu 13.5 mg/kg combined with ATRA 500 μg/d group (group D). Results Significant inhibition of tumor growth was shown in the group B, group C and group D as compared with group A (Ｐ＜0.01), with the tumor growth inhibition rate and the tumor weight inhibition rate at the 7 weeks after the treatment being 56.88%, 49.00%, 70.00% and 56.10%, 51.64%, 76.95%. Conclusion ATRA combined with 5-Fu can enhance the effects of 5-Fu in treating gastric carcinoma.
ObjectiveTo explore the conditions of synovial derived mesenchymal stem cells (SMSCs) differentiating into the fibrocartilage cells by using the orthogonal experiment. MethodsThe synovium was harvested from 5 adult New Zealand white rabbits, and SMSCs were separated by adherence method. The flow cytometry and multidirectional differentiation method were used to identify the SMSCs. The conditions were found from the preliminary experiment and literature review. The missing test was carried out to screen the conditions and then 12 conditions were used for the orthogonal experiment, including transforming growth factor β1 (TGF-β1), bone morphogenic protein 2 (BMP-2), dexamethasone (DEX), proline, ascorbic acid (ASA), pyruvic acid, insulin+transferrin+selenious acid pre-mixed solution (ITS), bovin serum albumin (BSA), basic fibroblast growth factor (bFGF), intermittent hydraulic pressure (IHP), bone morphogenic protein 7 (BMP-7), and insulin-like growth factor (IGF). The L60 (212) orthogonal experiment was designed using the SPSS 18.0 with 2 level conditions and the cells were induced to differentiate on the small intestinal submucosa (SIS)-3D scaffold. The CD151+/CD44+ cells were detected with the flow cytometry and then the differentiation rate was recorded. The immumohistochemical staining, cellular morphology, toluidine blue staining, and semi-quantitative RT-PCR examination for the gene expressions of sex determining region Y (SRY) -box 9 gene (Sox9), aggrecan gene (AGN), collagen type I gene (Col I), collagen type Ⅱ gene (Col Ⅱ), collagen type IX gene (Col IX) were used for result confirmation. The differentiation rate was calculated as the product of CD151/CD44+ cells and cells with Col I high expression. The grow curve was detected with the DNA abundance using the PicoGreen Assay. The visual observation and the variances analysis among the variable were used to evaluate the result of the orthogonal experiment, 1 level interaction was considered. The q-test and the least significant difference (LDS) were used for the variance analysis with a type Ⅲ calibration model. The test criteria (α) was 0.05. ResultsThe cells were certified as SMSCs, the double-time of the cells was 28 hours. During the differentiation into the fibrocartilage, the volume of the SIS-3D scaffold enlarged double every 5 days. The scaffolds were positively stained by toluidine blue at 14 days. The visual observation showed that high levels of TGF-β1 and BMP-7 were optimum for the differentiation, and BMP-7 showed the interaction with BMP-2. The conditions of DEX, ASA, ITS, transferrin, bFGF showed decreasing promotional function by degrees, and the model showed the perfect relevance. P value was 0.000 according to the variance analysis. The intercept analysis showed different independent variables brought about variant contribution; the TGF-β1, ASA, bFGF, IGF, and BMP-7 were more remarkable, which were similar to the visual observation. ConclusionIn the process of the SMSCs differentiation into the fibrocartilage, the concentrations of TGF-β1, ASA, bFGF, and IGF reasonably can improve the conversion rate of the fibrocartilage cells. The accurate conditions of the regulatory factor should be explored further.
【Abstract】 Objective To explore a method to identify the sensory and motor fascicles in peri pheral nervetrunk. Methods Thirty Wistar rats were selected to obtain whole spine. The spinal gangl ion, its dorsal root and ventral root,and sciatic nerve were harvested, Annexin V and Agrin specificities were observed with Western blot. In the experimental group,anterior branch and posterior branch of spinal nerve, sciatic nerve, and its muscular branch and cutaneous branch were harvested from 15 rats to make the observation of immunohistochemistry. In the other 15 rats, first antibody was replaced by PBS as control group. Different nerve fascicles were studied with Micro Raman scattering technique in 16 12-month-old New Zealand rabbits. Results The Annexin V and Agrin were special substances of sensory and motor nerves respectively and can act as specific antigens for identifying different nerve fascicles. There were significant differences in the intensity and breadth of the peak of the spectral properties between motor and sensory fascicles at frequencies of 1 088, 1 276, 1 439, 1 579 and 1 659 cm-1 .The peak intensity ratios of 1 276 to 1 439 cm-1 were 0.95±0.06 in motor nerve fascicles and 1.17±0.08 in sensory fascicles, showing significant differences (P lt; 0.05). Conclusion The Micro Raman spectra is more effective than immunohistochemistry in identifying different nerve fascicles, and it possesses as feasibil ity for cl inical appl ication.
ObjectiveTo isolate and identify the cartilage progenitor cells (CPCs) from normal cartilage, and to explore the influence of interleukin 1β (IL-1β) in different concentrations on its chondrogenesis. MethodsCPCs were isolated from normal cartilage of adult New Zealand white rabbit with the fibronectin adhesion assay;the cell phenotype was identified;and the cloning and differentiation of CPCs were observed. CPCs were incubated with H-DMEM in group A, with chondrogenic induced medium in group B, with chondrogenic induced medium+0.1 ng/mL IL-1β in group C and chondrogenic induced medium+1.0 ng/mL IL-1β in group D for 3 weeks. The histology, biochemistry, and real-time fluorescence quantitative PCR were performed to observe the effect of IL-1β on the chondrgenic differentiation. ResultsThe CPCs from normal cartilage expressed positively stem cell phenotype, which have similar ability of cloning and differentiation to stem cells. The cell pellets in groups C and D were significantly smaller than those in group B, and cell showed hypertrophic morphology change. There were more expressions of collagen type Ⅱ and collagen type X in group B than in group A, in group B than in groups C and D, and in group C than group D with Safranin O staining. The biochemistry results showed that collagen type Ⅱ content, glycosaminoglycan (GAG) content, and the ratio of GAG/DNA were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05);but the DNA content was significantly higher in groups C and D than in group B (P<0.05), and no significant difference between groups C and D (P>0.05). The real-time fluorescence quantitative PCR results showed that the relative mRNA expressions of collagen type Ⅱ, collagen type X, and Sox-9 were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05), but the relative mRNA expressions of Runx-2 and matrix metalloproteinase 13 were significantly higher in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05). ConclusionThere are CPCs having the character of stem cells in normal cartilage, and they have the capability of cloning and potential differentiation. IL-1β can inhibit the chondrogenesis of CPCs, and possibly promote the osteogenic differentiation.
Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.