Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokinesinduction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β1(TGF-β1,10 ng/ml), basic fibroblast growth factor(bFGF,25 ng/ml) addexamethasone (DEX,10-7mol/L) with 15%FBS in DMEM. Cartilage slices wereobtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as shortspindle shape, while the 5th MSCs showed longspindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positivestain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them（Pgt;0.05）, and there was significant difference between groups A and D（Plt;0.05）. No significant difference was noted in groups A,B and C on proliferation by MTT（Pgt;0.05）,except that of at the fourth day after transfection between groups A and C（Plt;0.05）. RT-PCR demonstrated that MSCs always had higher levelsof mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.
Objective To explore an effective method to culture and purify canine bladder transitional epithelial cells.Methods Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0.125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry. Results Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed micro filament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anticytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells.Different generations of cells showed diploid cells. Conclusion A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.
【Abstract】 Objective To explore an effective method to cultivate esophageal mucosa epithel ial cells (EMECs)of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Methods Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. Results The primary culture of canine EMECs arranged l ike slabstone. Immunohistochemical staining of CK-19 of the2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. E MECs werepolygon in shape and arranged l ike slabstone, and formed a single layer on the surface of SIS. The cells were contact ed closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, andshowed laminate arrangement. Conclusion With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6 %FBS is a simple and feasible method. SIS shows good biocompatibil ity and can be used as a good scaffold material in th e tissue engineered esophagus.
Objective To summarize the research progress of biological characteristics and advantages of Wharton’s jelly-mesenchymal stem cells (WJ-MSCs). Methods The related l iterature on the biological characteristics of WJ-MSCs,umbil ical cord blood MSCs (UBMSCs) and bone marrow MSCs (BMSCs) was extensively reviewed and analyzed. Results A large number of MSCs which are able to self-repl icate, self-renew and have high prol iferation and multipotent differentiation can be isolated from the Wharton’s jelly of umbil ical cord. WJ-MSCs have many advantages in isolation time, isolation efficience, expansion time, passage capacity, expansion capacity when compared with UBMSCs and BMSCs. Conclusion WJ- MSCs have numerous advantages of convenient and abundant sources, relatively pure, non-ethical issues, and so on, which can be used for cell transplant therapy, gene therapy, and the ideal seed cells of building tissue engineered organ, so they provide new ideas for tissue regeneration repair and reconstruction.
ObjectiveTo review the current progresses in purification strategies, biological characters, and the uses in tissue engineering of urine-derived stem cells (USCs). MethodsRecent relevant publications on the USCs were extensively reviewed, analyzed, and summarized. ResultsUSCs, usually isolated by adherence screening method, are a population of mesenchymal stem cells (MSCs)-like somatic stem cells possessing robust self-renew and multi-potential differentiation ability. Combined with using appropriate biomaterials and biological molecules, USCs can be used as a good cell source for tissue engineering. ConclusionAn alluring prospect exists on the USCs-related research. Further studies are required to investigate the origin, individual differences, and the therapeutic values of USCs.
ObjectiveTo summarize the research status and biological characteristics of stromal fibroblast in breast cancer. MethodsRelevant literatures about the breast cancer stromal fibroblasts published recently were collected and reviewed. ResultsIn addition to cancer cells, breast cancer included stromal cells. The fibroblasts were the major components of breast cancer stromal, which had significantly different biological characteristics from normal fibroblasts. The fibroblasts were characterized by α-SMA positive, p53 gene mutation, secretion of various cytokines or chemokines in addition to the production of collagen substances, involving in breast cancer growth, migration, invasion and metastasis through a variety of signaling pathways. ConclusionThe biological characteristics of stromal fibroblasts in breast cancer may reflect lesion properties, be of great importance to diagnosis and differential diagnosis and prognosis prediction of breast cancer. More attentions will be paid to the target therapy for stromal fibroblasts in breast cancer.
Objective To observe the effects of cryopreservation and resuscitation on the biological characteristics of mesenchymal stem cells (MSCs) derived f rom human umbilical cord blood. Methods MSCs were isolated and cultured f rom human umbilical cord blood in vitro. The cells were passaged , and the third generation of MSCs were cryopreserved in－196 ℃ liquid nitrogen for 4 weeks with cryopreservation medium , which contained 10 % dimethyl sulfoxide (DMSO) and 90 % fetal calf serum ( FCS) . The morphology , proliferation and differentiation of MSCs were investigated and compared with those of MSCs before cryopreservation. Results There was no significant difference of morphology between pre-cryopreserved MSCs and the ones af ter resuscitation. It was observed that all MSCs were spindle-shaped and showed adherence growth characteristic before and af ter cryopreservation. The cell growth curves of MSCs were also similar before and af ter cryopreservation. Even though the curve of resuscitated MSCs descended a little as compared with that of pre-cryopreserved MSCs , there was no significant difference ( Pgt; 0. 05) . After 2-week adipocytic differentiation induction , fat drops could be found in the kytoplasm of MSCs and they were red when stained with oil-red O staining , which suggested that MSCs could be induced and differentiated into adipocytes. Af ter 4-week osteoblastic differentiation induction , MSCs could be induced and differentiated into osteoblasts , and calcium node showed black when stained with Von Kossa staining. There were no significant changes of the differentiating ability of MSCs into adipocyte and osteoblast before and after cryopreservation. Conclusion MSCs derived from human umbilical cord blood maintains their biological characteristics af ter cryopreservation and resuscitation.
ObjectiveTo summary the relationship between CD147 and the occurrence and development of hepatocellular carcinoma, and its roles in clinical diagnosis and treatment. MethodReferring to the related literatures in recent years at home and abroad, the concept of CD147, and its relationship with the occurrence, development, invasion, and metastasis of hepatocellular carcinoma were reviewed. ResultsCD147 plays a key role in the development, progress, invasion, and metastasis of hepatocellular carcinoma. CD147 can be used as a long-term outcome predictor for hepatocellular carcinoma patients and also began to show its in hepatocellular carcinoma target therapy. ConclusionThere are numerous studies about the relationship between CD147 and hepatocellular carcinoma, but still exists some problems to be further studied.
ObjectiveTo investigate the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in microenvironment of premature senescence of nucleus pulposus cells (NPCs) so as to lay a foundation for the repair of intervertebral disc degeneration by BMSCs transplantation. MethodsHuman degenerative nucleus pulposus and normal bone marrow were collected, and then NPCs and BMSCs were isolated, cultured, and identified. The 3rd passage BMSCs and the 1st passage NPCs with premature senescence were co-cultured without contact in the Transwell culture system. NPCs to BMSCs ratio was 75%:25% (group A), 50%:50% (group B), and 0:100% (group C). The morphological changes of BMSCs were observed by inverted phase contrast microscopy and transmission electron microscopy. At 3 and 6 days after co-culture, cell counting kit 8 was used to detect cell viability, flow cytometry was used to observe the cell cycle and detect DNA metabolism after BrdU labeling. Cell senescence was also evaluated by detecting senescence associated β-galactosidase (SA-β-gal) activity. ResultsThe typical morphology of cell senescence was seen in groups A and B, especially in group A. At 3 and 6 days after co-culture, the cell survival rate of group A was significantly lower than that of group B (P<0.05). At 3 days after co-culture, the proportion of cells in G1 phase in group A was significantly higher than that in groups B and C (P<0.05), the proportion of cells in S phase in group A was significantly lower than that in groups B and C (P<0.05). At 6 days, the proportion of cells in G1 phase in group A was about 81.0%, and the proportion of cells in S phase and G2 phase decreased, showing significant difference when compared with groups B and C (P<0.05); the proportion of cells in G1 phase in group B was about 74.4%, showing significant difference when compared with group C (P<0.05). BrdU content in group A was significantly lower than that in groups B and C at 3 and 6 days after co-culture (P<0.05), but no significant difference was found between groups B and C at 3 days (P>0.05); Brdu content in group B was also significantly reduced when compared with group C (P<0.05) at 6 days. At 6 days, SA-β-gal activity was significantly increased in groups A and B, and significant difference was shown in SA-β-gal positive cell number between groups (P <0.05). ConclusionPremature senescence of NPCs can down-regulate the proliferation capacity of co-cultured BMSCs by the paracrine effect. The greater proportion of NPCs with premature senescence is, the earlier senescence of BMSCs will be induced.
Following the peritendon was removed by means of microsurgical technique, the tenocyte was isolated from the human embryonic tendons by digesting it with trypsin and collagenase. These cells were all stored in frozen condition until they were cultured by F12 culture fluid added with 20% FBS to the 15th generation.These cells were able to grow adhering to the wall and stop growing with contact inhibition. The time of cellsgroup duplication was 4 days, which was similar to the peak time of its mitosis. The number of its chromosome group 2n=46 was 87.5-91.0%. The optimal conditions for tendon cell culture in vitro were investigated, and it was found that after they were reaminated and subcultured the frozen storage didn’t influence their growth, morphology, genetic characteristics. In our research we detected the content generation cells and found the cultured human embryonic tenocyte had same ability never changed with the cells subcultured. We also disscussed the future of tenocyte-a biomaterial in the field of artificial implant.