OBJECTIVETo study the overexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells for repairing articular cartilage injury in vivo.

METHODSRabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with lentivirus vector containing Sox9 gene and then cartilage specific molecule was detected by RT-PCR in vitro. Total 48 knee joints of 24 mature New Zealand white rabbits were randomly divided into 3 groups according to different defect treatment. After animals anesthesia,a full-thickness cylindrical cartilage defect of 4 mm diameter and 3 mm deep was created in the patellar groove using a stainlesssteel punch. Meanwhile, the transfected cells were implanted to repair the rabbit model with full-thickness cartilage defects. Cartilage defects tissue was observed with light microscope, electron microscope, HE and immunohistochemistry staining to assess the repair of defects by the complex at 6 weeks or 12 weeks after the implantation.

RESULTSAt 3 days after the transfection, Sox9 gene expression was highest and Sox9 gene expression decreased with the increase of time. At 3 days after the transfection, the expression of collagen type II began and reached the peak at 14 days. It showed that the bone marrow mesenchymal stem cells went into chondrogenic differentiation after transfected by Sox9 gene. Histological observation showed that at 6 weeks after the operation, the defects in the experimental group was filled with hyaline like cartilage tissue, 12 weeks after operation,the defects of cartilage and subchondral bone had satisfactory healing. Both at 6 and 12 weeks postoperatively, the defects were filled with fibrous tissues in control groups. Meanwhile, immunohistochemical staining of sections with type II collagen antibodies showed the proteins in the regenerated tissue stained positive for type II collagen and stronger than the control groups. The histological scoring system indicated that the cartilage repair of experiment groups were better than the two control groups with statistical significances.

CONCLUSIONOverexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells (BMSCs) promote the repair of cartilage defect.

Animals ; Bone Marrow Cells ; metabolism ; Bone Marrow Transplantation ; Cartilage, Articular ; injuries ; metabolism ; Cell- and Tissue-Based Therapy ; Female ; Genetic Vectors ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; metabolism ; Osteoarthritis ; genetics ; metabolism ; therapy ; Rabbits ; SOX9 Transcription Factor ; genetics ; metabolism ; Tissue Engineering

Adult ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Glomerulonephritis ; complications ; Humans ; Kidney Failure, Chronic ; drug therapy ; Male ; Middle Aged ; Phytotherapy ; Powders

OBJECTIVETo study the inhibition effect of CyclinD1 specific small interfering RNA(siRNA) on CyclinD1 gene expression in human keloid fibroblast, investigating the effect of CyclinD1 specific siRNA (siRNA-CyclinD1) on the cell cycle, multiplication and apoptosis.

METHODSAccording to the principle of siRNA design, siRNA-CyclinD1 was designed and the keloid fibroblast were transfected. RT-PCR was used to examine CyclinD1 mRNA expression. Flow cytometry was used to examine cell cycle. The apoptotic rate was analyzed by using Annexin V-FITC/PI kit. The DNA gragmentation were measured by DNA ladder analysis.

RESULTSAfter transfection, the expression of CyclinD1 mRNA decreased remarkably. Twenty-four, forty-eight and seventy-two hours after transfection, the radio of G1 stage cell was (59.80 +/- 3.06)%, (66.01 +/- 4.03)% and (67.43 +/- 5.35)%, all significantly higher than in the control group (54.50 +/- 5.35)%; the radio of S stage cell was (18.40 +/- 1.42)%, (17.21 +/- 1.76)% and (11.07 +/- 1.00)%, significantly lower than in the control group (22.33 +/- 1.49)%; the proportion of the cells in G1 stage increased and those in the S stage decreased in the keloid fibroblast transfected with siRNA-CyclinD1. The apoptotic rate of the siRNA-CyclinD1 group was (7.82 +/- 0.45)%, (15.71 +/- 1.06)%, (18.32 +/- 1.08)%, all significantly higher than in the control group (0.68 +/- 0.12)%, and the DNA gragmentation can be seen remarkably.

CONCLUSIONSChemically synthesized siRNA- CyclinD1 effectively inhibits. The expression of CyclinD1 in keloid fibroblast thus arresting the cell cycle at G1 stage and enhancing cell apoptosis. Our study provided a preliminary results in seaching of a RNAi therapy of keloid.

Apoptosis ; genetics ; Cells, Cultured ; Cyclin D1 ; genetics ; Fibroblasts ; cytology ; metabolism ; Flow Cytometry ; Gene Silencing ; Humans ; Keloid ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; Transfection

Neurogranin, a neuron-specific postsynaptic protein, has been considered to play an important role in synaptic plasticity and learning and memory. The present study aimed to investigate the effects of prenatal restraint stress on neurogranin expression in rat offspring hippocampus. Pregnant rats were given a restraint stress (3 times a day for 7 d, 45 min each time) at the late stage of gestation except that in the control group. The offspring rats were divided into four groups: female control group, male control group, female stress group and male stress group. Expression of neurogranin was determined by immunohistochemistry and Western blot. The results showed that neurogranin-positive immunostaining was detected in all areas of the hippocampus. The staining density was stronger in the CA1 and CA3 regions than that in the dentate gyrus (DG) region. Western blot assay showed that neurogranin protein level in female and male prenatal stressed offspring was significantly lower than that in the controls (P<0.01). Neurogranin level was significantly lower in the female stress group than that in the male stress group, whereas there was no significant gender difference in the control group. Immunohistochemical data further confirmed these results. The present study provides evidence that prenatal restraint stress induces gender-dependent decrease in neurogranin expression in the offspring hippocampus. The prenatal restraint stress-induced decrease in neurogranin expression in the hippocampus might be associated with the deficit in spatial learning and memory reported previously.
Animals ; Blotting, Western ; Female ; Hippocampus ; chemistry ; Immunohistochemistry ; Male ; Neurogranin ; analysis ; Pregnancy ; Prenatal Exposure Delayed Effects ; Rats ; Rats, Sprague-Dawley ; Restraint, Physical ; Stress, Psychological ; metabolism

BACKGROUNDHypoxia-inducible factor-1alpha (HIF-1alpha) is a transcriptional factor that could improve the stimulation of angiogenesis and the metabolic adaptation of tumor cells to hypoxia. A recent study showed that HIF-1alpha could induce colon cancer cells epithelial-mesenchymal transition (EMT). However, no evidence indicates a similar correlation in human prostate cancer cells. This study was designed to evaluate the effect of HIF-1alpha over-expression on the EMT in human prostate cancer cells.

METHODSWe selected the appropriate cell line for HIF-1alpha induction from those EMT negative prostate cell lines through vimentin gene detection by RT-PCR. As the result, LNCaP cell line is the best one for further experiment. LNCaP cells were transfected with recombinant plasmid pcDNA3.1 (-)/HIF-1alpha and pcDNA3.1 (-) control vector by Lipofectamine 2000 system. The positive cell colonies were confirmed by indirect immunofluorescence labeling. Then Transwell polycarbonate filter was used to analyze the invasive potency. The expression of EMT associated proteins, E-cadherin and vimentin, was detected by Western blotting.

RESULTSAmong four of the EMT negative cell lines, LNCaP was the only one expressed the vimentin gene but not the associated protein. The expression level of HIF-1alpha in LNCaP/HIF-1alpha was distinctly higher than that in LNCaP/pcDNA3.1 and LNCaP. The cell numbers of LNCaP/HIF-1alpha that penetrated through the Transwell filter were higher than that of LNCaP/pcDNA3.1 and LNCaP. Compared with the LNCaP/pcDNA3.1 and LNCaP cells, the expression of vimentin was up-regulated in LNCaP/HIF-1alpha, whereas the expression of E-cadherin was down-regulated.

CONCLUSIONSOver-expression of HIF-1alpha stimulates the invasion potency of human prostate carcinoma cells through EMT pathway. The expression of E-cadherin and vimentin, playing established roles in EMT, could be regulated by HIF-1alpha in human prostate cancer cell line.

Cadherins ; genetics ; Cell Line, Tumor ; Epithelial Cells ; chemistry ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; physiology ; Male ; Mesoderm ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Messenger ; analysis ; Vimentin ; genetics

OBJECTIVESTo evaluate the application of two-dimensional electrophoresis in the research of differentially expressed proteins in human spermatozoa.

METHODSTwo-dimensional gel electrophoresis was performed on 4 sperm samples from normal healthy men and another 4 from asthenospermia patients. After silver staining, the differential expression proteins were analyzed by PDQuest 2D analysis software.

RESULTSSeven differential protein spots were identified, 2 expressed highly in the asthenospermia sperm but lowly in the normal spermatozoa, while the other 5 expressed just the opposite way.

CONCLUSIONThe protein profiles of differential expression between the normal spermatozoa and idiopathic asthenospermia were established and some differential proteins were found. The data obtained in this study may help prepare the ground for further studies on the isolation and identification of differentially expressed proteins in human asthenospermia sperm.

Case-Control Studies ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Male ; Oligospermia ; metabolism ; Seminal Plasma Proteins ; analysis ; biosynthesis ; Spermatozoa ; chemistry

BACKGROUNDThymokidney has been reported as an approach for a vascularized thymus for transplantation to induce donor specific tolerance. A completely thymectomized model which ensures that the obtained thymus is not injured has not been developed yet and it would be useful for evaluating autologous thymokidney function in rats.

METHODSAdult Sprague-Dawley male rats weighing 150 - 300 g (n = 30) underwent non-invasive intubation with the assistance of an improved self-made wedge-shaped cannula made from a 2-ml plastic syringe and transillumination from the anterior tracheal area by an operation spotlight. The rats then received a thoracotomy while their breathing was supported by a small animal ventilator, and both lobes of the thymus were entirely extirpated under a 10× microscope. The postoperative survival rate of the rats was recorded, and changes in the T-cell reservoir from 9 of 30 rats within 21 days after surgery were monitored using flow cytometry. The complete thymectomy rate was confirmed by autopsy and histological examination on 21 days post-operation.

RESULTSThe postoperative survival rate of rats was 100%. The exsected thymus was free of injury and the rate of complete thymectomy was 100%.

CONCLUSIONSThis model has a stable survival rate and complete thymectomy is able to be achieved. The obtained thymus tissue is free of injury and can be used for transplantation.

Animals ; Intubation, Intratracheal ; methods ; Male ; Rats ; Rats, Sprague-Dawley ; Thoracotomy ; methods ; Thymectomy ; methods ; Thymus Gland ; surgery

OBJECTIVETo observe the expression profiles between two metastasis-associated proteins in different prostate cancer cell lines and explore the molecular mechanisms of bone metastatic potentials.

METHODSExpressions of E-cadherin and vimentin in two prostate cancer cell lines (LNCaP and IA8) with different metastatic potentials were detected by Western blotting.

RESULTSThere was remarkable difference in the expressions of E-cadherin and vimentin between the highly metastatic cell line and the lowly one. As one of the adhesion associated proteins, E-cadherin was detected with high level of expression in LNCaP cell line, which was well known as low metastatic potential. However, E-cadherin did not expressed in IA8 with high metastatic potential. And as one of the cytoskeleton proteins, vimentin expression was high in IA8, but not in LNCaP.

CONCLUSIONThere is definitely difference in the metastatic phenotypes (E-cadherin and vimentin) among cell lines with different metastatic potentials. The expressions of E-cadherin and vimentin proteins may play important roles in promoting and inhibiting the metastasis of prostate cancer respectively, and may be considered to be valuable in evaluating the malignant degree, predictable metastasis and prognosis of prostate cancers.

Cadherins ; biosynthesis ; Cell Line, Tumor ; Humans ; Male ; Neoplasm Metastasis ; Prognosis ; Prostatic Neoplasms ; metabolism ; pathology ; Vimentin ; biosynthesis

OBJECTIVETo determine the characteristics of epithelial-mesenchymal transition (EMT) in different human prostate cancer cell lines and explore the molecular mechanisms of bone metastatic potentials.

METHODSExpressions of E-cadherin, N-cadherin and Vimentin in several prostate cancer cell lines (LNCaP, C4, C4-2, IF11, IA8, Du145 and PC-3) with different metastatic potentials were detected by Western blotting.

RESULTSThere was remarkable difference in the expressions of E-cadherin, N-cadherin and Vimentin between these cell lines. As one of the adhesion associated proteins, E-cadherin was detected with high expression in LNCaP, C4, C4-2 and PC-3, whereas with a low expression in IF11, IA8 and Du145. However, as one of the mesenchymal proteins, N-cadherin was shown to be completely different from Vimentin expression profile in these cell lines.

CONCLUSIONThere is actual difference in the EMT phenotypes among cell lines with different metastatic potentials. LNCaP, C4, C4-2 and PC-3 are cells without EMT change, while IF11, IA8 and Du145 are positive for EMT. The expressions of EMT associated proteins play important roles in promoting and repressing the metastasis of prostate cancer.

Blotting, Western ; Bone Neoplasms ; secondary ; Cadherins ; biosynthesis ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; Epithelial Cells ; pathology ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Vimentin ; biosynthesis

OBJECTIVECanine model established for tracheal defect reconstruction, to investigate the outcome of tracheal reconstruction with combination of polypropylene and flap.

METHODSAbout 3.5 to 4 centimeter cervical trachea was resected and replaced with artificial trachea made from monofilament knitted polypropylene and surgical flap. Covered stent was implanted postoperatively. Survival period and quality of life were recorded, bronchofibroscopy, X-ray films and HE sections were performed.

RESULTSSix dogs survived well and another two died. The causes of death were respiratory failure in 1 and infection in another. Stenosis of anastomosis in 1 was recorded during survival period. The dogs started drinking and eating on the second postoperative day, no dyspnea was found. The animals were sacrificed at 2, 4, 8 weeks and 6 months after surgery. Soft tissue growth was found in polypropylene net 2 weeks after surgery and more at 4 weeks. The polypropylene net was covered completely with soft tissue at 8 weeks and 6 months postoperatively, the hardness and sustentation degree were enhanced following the growth and fibrosis of soft tissue. The squamous epithelium and columnar epithelium were observed healing well by HE staining method.

CONCLUSIONSOne-stage operative artificial trachea made from monofilament knitted polypropylene which has good histocompatibility and surgical flap is the closer artificial trachea to native trachea. It has a promising prospect in clinical use.

Animals ; Dogs ; Polypropylenes ; Prostheses and Implants ; Reconstructive Surgical Procedures ; instrumentation ; methods ; Skin Transplantation ; Surgical Flaps ; Trachea ; surgery