Objective To analyze the changes of gene expression profiles during the process that human bonemarrow mesenchymal stem cells (hBMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine (5-aza). Methods hBMSCs were isolated from marrow of obsolete ribs and induced with 5-aza. Then immunocytochemicalstaining was used to detect the expressions of α-actin, cardiac troponin T (cTnT), and connexin 43, and the percentage ofcTnT positive cells was tested with flow cytometry. In the process of differentiation, variation of gene expression was screenedwith Genechi ps Operating System of human gene expression profiles. And the differentially expressed genes were functionallyanalyzed and hierarchical clustered. Results When BMSCs were induced in vitro with 5-aza, part of the cells turnedinto myogenic cells morphologically. Before induction, immunocytochemical staining for α-actin and cTnT showed sl ightpositive and for connexin 43 showed negative. While after 3 weeks of induction, immunocytochemical staining for α-actin,cTnT, and connexin 43 showed all positive. With flow cytometry, the percentage of cTnT positive cells was 7.43% ± 0.02%before induction, but it was 49.64% ± 0.05% after induction. During differentiation, 1 814 differentially expressed geneswere reported by gene chi ps. Of them, 647 genes were divided into 5 groups with hierarchical clustering. They had variousbiological functions, involving signal transduction, cell metabol ism, prol iferation, differentiation, development, andtopogenesis. Conclusion hBMSCs can differentiate into cardiomyogenic cells with the induction of 5-aza in vitro. Multi plegenes related with signal transduction, transcri ption, and growth factors are involved during this process.
Because of the diversity and complexity of clinical indicators, it is difficult to establish a comprehensive and reliable prediction model for induction of labor (IOL) outcomes with existing methods. This study aims to analyze the clinical indicators related to IOL and to develop and evaluate a prediction model based on a small-sample of data. The study population consisted of a total of 90 pregnant women who underwent IOL between February 2023 and January 2024 at the Shanghai First Maternity and Infant Healthcare Hospital, and a total of 52 clinical indicators were recorded. Maximal information coefficient (MIC) was used to select features for clinical indicators to reduce the risk of overfitting caused by high-dimensional features. Then, based on the features selected by MIC, the support vector machine (SVM) model based on small samples was compared and analyzed with the fully connected neural network (FCNN) model based on large samples in deep learning, and the receiver operating characteristic (ROC) curve was given. By calculating the MIC score, the final feature dimension was reduced from 55 to 15, and the area under curve (AUC) of the SVM model was improved from 0.872 before feature selection to 0.923. Model comparison results showed that SVM had better prediction performance than FCNN. This study demonstrates that SVM successfully predicted IOL outcomes, and the MIC feature selection effectively improves the model’s generalization ability, making the prediction results more stable. This study provides a reliable method for predicting the outcome of induced labor with potential clinical applications.
Objective The biological treatment of intervertebral disc degeneration becomes a research hotspot in recentyears. It is necessary to find an effective approach to induce bone marrow mesenchymal stem cells (BMSCs) differentiate to disc cells which could make appl ication of cell transplantation as a treatment of intervertebral disc degeneration. To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit BMSCs into nucleus pulposus-l ike cells. Methods The eukaryotic expression vector of pcDNA3.1IE-SOX9Flag was constructed. Rabbit BMSCs were isolated and cultured from one-month-old New Zealand white rabbits and were induced into osteogenetic cells in the osteogenesis supplement medium; and the cell surface markers were detected by flow cytometry. The cells at the 3rd passage were randomly divided into 3 groups: in transfected group, the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag; in negative control group, the cells were transfected with plasmid pcDNA3.1; and in blank control group, the cells were treated with the media without recombinant plasmid. After selected by G418 for 7 days, the cells were harvested and RT-PCR was employed to assay SOX9 mRNA and collagen type II gene (Col2al) mRNA expressions in BMSCs. The expression of SOX9 protein was assayed by Western blot and collagen type II expression was also observed by immunohistochemical staining. Results The SOX9 eukaryotic expression vector was constructed successfully. The BMSCs after 5 days of osteogenetic induction were positive for the alkal ine phosphatase staining. What was more, CD44 expression was positive but CD34 and CD45 expressions were negative. The transfection efficiency was 34.32% ± 1.75% at 72 hours after transfection. After 2 weeks of transfection, BMSCs turned to polygonal and ell iptical. And the cell prol iferation was gradually slow which was similar to the growth characteristic of nucleus pulposus cells. RT-PCR identification showed that SOX9 mRNA and Col2al mRNA expressions were positive in transfected group, and were negative in 2 control groups. Western blot detection showed that SOX9 protein expressed in transfected group but did not express in the control groups. At 2 weeks after transfection, the result of the immunohistochemicalstaining for collagen type II was positive in transfected group. Conclusion The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rabbit BMSCs, the transfected BMSCs can differentiate into nucleus pulposus-l ike cells, which lays a theoretical foundation for treatment of intervertebral disc degeneration with BMSCs transplantation.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
Objective To review the research progress of the current methods of inducing bone marrow mesenchymal stem cells (BMSCs) to chondrogenic differentiation in vitro so as to provide references for researches in cartilage tissue engineering. Methods Various methods of inducing BMSCs differentiation into the chondrogenic l ineage in vitro inrecent years were extensively reviewed and analyzed. Results Adding exogenous growth factors is still the mainly methodof inducing BMSCs differentiation into the chondrogenic l ineage; among the members, transforming growth factor β (TGF-β) family is recognized as the most important chondrogenic induction factor. Other important inducing factors include various chemical factors, physical factors, transgenic methods, and the microenvironmental induction. But the problems of low inducing efficiency and unstable inducing effects still exist. Conclusion The progress of chondrogenic induction of BMSCs promotes its util ization in cartilage tissue engineering. Further researches are needed for establ ishing more efficient, simpler, and safer inducing methods.
Objectives To evaluate the clinical effectiveness and safety of combined induction therapy of interferon (IFN) with chemotherapy for survival of the patients with advanced non-small cell lung cancer (NSCLC) by meta-analysis. Methods All clinical trials of addition of IFN plus chemotherapy versus chemotherapy alone for induction therapy to advanced NSCLC patients in MEDLINE (1966-2006), EMBASE (1984-2006.1) and The Cochrane Library (Issue 1,2006) were identified. The references of related studies and Education Books of ASCO and ESMO meeting were handsearched. The quality of included trials was evaluated. Data were extracted by two reviewers independently with a designed extraction form. RevMan 4.2.7 software was used for data analysis. Results Five randomized controlled trials involving 360 patients were included. The pooled result of 3 studies showed that IFN plus chemotherapy induction treatment did not improve 1-year survival rate with RR 0.76, 95%CI 0.46 to 1.26. The pooled result of 5 studies showed that IFN plus chemotherapy induction treatment did not improve response rate with RR 1.40, (0.83 2.34). The pooled result showed that IFN plus chemotherapy induction treatment might significantly increase leukopenia and thrombocytopenia with RR 2.61,95%CI1.70 to 3.99) and RR 4.78,95%CI 1.87 to 12.19 respectively . Conclusion Insufficient data exists to state whether IFN plus chemotherapy induction treatment can improve 1-year survival rate and response rate. IFN plus chemotherapy may increase occurrence of leucopenia and thrombocytopenia. Further studies are warranted.
Objective To investigate the effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindl imb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologousPRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, al izarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 (Cbfα1) of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was (3.06 ± 0.46) × 105/μL and (18.08 ± 2.10) × 105/μL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P lt; 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P lt; 0.05). Al izarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineral ization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineral ization rate (P lt; 0.05), the experimental group at 12.50% had a higher mineral ization rate than other experimental groups (P lt; 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hoursafter culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.
Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.
Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
The optimal treatment of stage ⅢA-N2 non-small cell lung cancer （NSCLC） remains controversial. Resultsof primary surgery alone are not satisfied. Surgery after induction chemotherapy yields better outcomes compared to resectiononly which has been widely accepted. Randomized studies show induction chemotherapy followed by either radiotherapy or surgery have approximately equivalent survival outcomes，significant improved survival can be achieved by combined surgery in selected patients. Low-grade N2，effective response and mediastinal downstaging after induction therapy，and successful complete resection by lobectomy，are good indications of surgery. Ideal treatments are approached base on theheterogeneity of N2 . Patients with bulky or fixed N2 disease should be considered for radical chemo-radiotherapy，and surgeryshould be a part of multi-modality management for patients with non-fixed，non-bulky，single-zone N2 disease. Further randomized trials of surgery added to multi-modality management in patients with multi-zone N2 disease should be taken in order to establish possible subgroups of patients might be benefitted more from the addition of surgery.