Construction and expression of growth differentiation factor-5 gene prokaryotic expression plasmid and its cartilage induction activity in vivo / 中国组织工程研究

BACKGROUND: Growth differentiation factor-5 (GDF-5) plays an important role in the development and formation of cartilage, extremities, and joints, which is a widely used joint development marker.OBJECTIVE: To express mature peptide of human GDF-5 in E. coil by the way of genetic engineering, and to explore the inductive activity of recombinant protein in vivo.DESIGN, TIME AND SETTING: The observation experiment based on gene was performed at the Analysis and Testing Center of Southern Medical University from January to June 2006.MATERIALS: Human fetus cartilage tissue was harvested from Department of Gynaecology and Obstetrics, and the consent was obtained from the family. Ten KM mice were purchased from experimental animal center of Southern Medical University, half male and half female, weighing 18-22 g, aged 6-8 weeks.METHODS: The hGDF-5 gene encoding mature peptide was gained by RT-PCR from the total RNA which was extracted from fetus cartilage tissues, and was inserted into the pET22b(+) vector to construct recombinant prokaryotic expression plasmid pET22b(+)-GDF5, which was transformed into E. coil BL-21 to be expressed after IPTG induction. Proteins of interest were purified with sepharose chelated with nickel ions (Ni2+) and then implanted in mouse hindlimb muscle to evaluate the biological activities by routine hematoxylin-eosin staining.MAIN OUTCOME MEASURES: The expression, sequencing of target gene was observed by agarose gel electrophoresis, and the protein expression was detected by SDS-PAGE electrophoresis, meanwhile, the GDF5-inducing activity was evaluate by histological observation.RESULTS: RT-PCR product was about 350 bp in length, which was confirmed by double enzyme digestion of the recombinant plasmid, sequencing result was in agreement with the reported hGDF-5 sequence in Genbank. SDS-PAGE analysis showed a conspicuous band representing a new foreign protein with relative molecular mass of approximately 14 KD after induced expressioin. Cartilage tissues were formed in the mouse muscle where the purified proteins were implanted. CONCLUSION: The integral human GDF-5 mature peptide gene was cloned successfully from human fetus cartilage tissue and a high-yield expression was achieved in E. coli, the pudfied protein has chondrogenic activities in vivo.

The committed differentiation of bone marrow mesenchymal stem cells into tenocytes / 解放军医学杂志

Objective To investigate the feasibility of committed differentiation of rhesus bone marrow mesenchymal stem cells (MSCs) into tenocytes induced by BMP12. Methods MSCs were transfected with pTARGETTM bearing BMP12 gene by electroporation. The transfected cells were identified by morphological observation and molecular biological measure. Results Under the observation of light microscope, the morphological features of transfected cells changed significantly compared to parental MSCs. RT-PCR data showed the transfected cells had the mRNA expression of BMP12 and collagen Ⅰ, but without that of collagen Ⅲ. 98.39% of the transfected cells were CD44+ and negative for HLA-DR. Conclusion BMP12 could induce MSCs into tenocytes, and bone marrow MSCs might be the optional seed cells for tendon tissue engineering.

Expression of collagen subtype I mRNA during autogenous tendon formation induced by grafted human hair keratin artificial tendon / 中国组织工程研究

BACKGROUND: Numerous experiments and clinical practice show that human hair keratin artificial tendon induces the organism to form autogenous tendon. The process of autogenous tendon formation mainly involves the synthesis, secretion and package of collagen subtype I.OBJECTIVE: To explore the role of collagen subtype I mRNA expression in autologous tendon formation after human hair keratin artificial tendon implantation.DESIGN: A completely randomized controlled experiment based on the experimental animals.SETTING: Department of Histology and Embryology and Department of Biochemistry and Molecular Biology of Southern Medical University.MATERIALS: The experiment was conducted in the Experimental Animal Center of the First Military Medical University of Chinese PLA from May 2003 to September 2004. Totally 33 New Zealand rabbits of either gender,weighing 2.0 to 2. 5 kg, were provided by the center. The animals were randomly divided into experiment 3, 6, 9, 12, 16 and 20 weeks groups, negative control 9 and 20 weeks groups and normal control group. Among them,experiment 3 and 6 weeks group and normal control group had 3 rabbits in each and the other groups had 4 rabbits. Human hair keratin artificial tendons were normal human hair treated by a series of biochemical methods and were supplied by the Department of Biochemistry & Molecular Biology of the university. The human hair keratin artificial tendons were divided into three groups with different degradation rates, namely, fast(F), medium(B) and slow(Z). The tendons were made up of the fast, medium and slow degradation groups mixed at the ratio of 4: 3: 3.off by 1.0- 2.0 cm, human hair keratin artificial tendon was grafted by end-to-end anastomosis with both ends of the broken tendon before sewing control group, no artificial tendon was implanted although the animals ungroup was normal rabbits' tendon. Sampling was carried out at 3, 6, 9, 12, 16and 20 weeks after human hair keratin artificial tendon implantation in experiment groups, and at 9 and 20 weeks after operation in negative control group, respectively. The expression of collagen subtype I mRNA was detected at weeks 3, 6, 9, 12, 16 and 20 after grafting using reverse transcription polymerase chain reaction technique.MAIN OUTCOME MEASURES: The ratio of collagen subtype I mRNA to Glyceraldehyde-3-phosphate dehydrogenase(GADPH) mRNA in normal tendon and autogenous tendon induced by human hair keratin artificial tendon at all time points was calculated, and significance test between all these paired groups were performed.collagen subtype I mRNA/GADPH mRNA expression was 0.96 ±0.02 in expression of collagen subtype I mRNA/GADPH mRNA in autogenous tendon induced by human hair keratin artificial tendon in experiment group appeared at week 3, increased rapidly at week 3 to 6, peaked at week 6, and remained stable at week 9 to 20. The expression at week 6 was significantly higher in experiment group than in normal control group( F = 6. 254, P ＜ 0.05); the expression at other weeks was also significantly higher in experiment group than in normal control group( F= 1. 258 - 1. 987, P ＞ 0.05).CONCLUSION: The activation, proliferation and secretion of collagen protein as well as the synthesis of collagen subtype I by tenocytes may be responsible for autologous tendon formation after human hair keratin artificial tendon implantation.

Cloning of integral mature peptide gene of human GDF-5 / 华中科技大学学报（医学）（英德文版）

The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.

Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.</p><p><b>METHODS</b>Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with alpha-(32)P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by Atlas Image analysis software Version 1.01a.</p><p><b>RESULTS</b>Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.</p><p><b>CONCLUSION</b>The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.</p>

Culture and pluripotentiality of human marrow mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To cultivate human mesenchymal stem cells (MSC) derived from fetal bone marrow and examine their pluripotentiality.</p><p><b>METHODS</b>Bone marrow mononuclear cells from human fetal femur were collected by Percoll gradient centrifugation. The low density cells including MSCs were cultivated and expanded in MSCGM media. The characteristics of the multipotent MSCs were observed by implanting them into nude mice for 4 weeks.</p><p><b>RESULTS</b>Human fetal marrow MSCs were successfully cultivated and differentiated in vivo into many kinds of tissues such as bone, cartilage, adipose, skeletal muscle and tendon-like tissue.</p><p><b>CONCLUSION</b>Human fetal marrow MSCs were multipotent stem cells.</p>

Inhibition of resveratral on lipid peroxidative reaction and reactive oxygen species of early experimental rat spinal cord injury / 中国临床药理学与治疗学

AIM: To study the effects of resveratral on lipid peroxidative reaction and reactive oxygen species (ROS) in experimental spinal cord injury rats. METHODS: The weight dropping method was used to produce the experimental spinal cord injury (SCI) in adult rats. Resveratrol (Res) and methylprednisolone (MPSS) were given intraperitoneally by the bolus injections of 50, 100 mg?kg -1 and 100 mg?kg -1 , respectively, immediately after induction of SCI. And then the effects of Res were observed at 1, 24, 48 h after SCI compared with MPSS. RESULTS: Res could obvious prevented reduction of superoxide dismutase (SOD) activity and reduce malondialdehyde (MDA) production and inhibit the ROS level in the injured spinal cord tissue in comparison with the SCI model at the dose of 50 mg?kg -1 and 100 mg?kg -1 , which the most remarkable effects was at 48 h with the inhibition rate over 40%. The effects of Res were the same as or even greater than those of MPSS. CONCLUSION: Res may effectively protect the spinal cord from lipid peroxidative reaction and ROS damage in SCI.

CYTOCHEMISTRY OF LYSOSOMAL ENZYMES AND PRIMARY LYSOSOMES / 解剖学报

Lysosomes are organelles that extensively exist in cells of animals and plants. The outer boundary of lysosomes is the unit membrane that surrounds high electron dense substances. The chief components of lysosomes are a variety of hydrolases that can hydrolyze many intracellular components including macromolcular substances produced by cell itself and organic substances that come from extracellular environment. Therefore, the function of lysosomes is to degrade exogenous materials (heterophagics) and endogenous materials (autophagies). Moreover, lysosomse play an important role in the physiologic, pathlogic, pharmacologic and immunologic processes. The present review describes the enzymes within the lysosomes and explains the morphology and function of the primary lysosomes.

Effects of resveratrol on secondary spinal cord edema and activity of lactic dehydrogenase and ATPase in experimental spinal cord injury of rats / 中国药理学通报

AIM To study the effects of resveratrol on secondary spinal cord edema and the activity of lactic dehydrogenase and Na +, K + ATPase in experimental spinal cord injury (SCI)rat. METHODS The weight dropping method was used to produce the experimental spinal cord injury in adult rats. Resveratrol (Res) and methylprednisolone (MPSS) were intraperitoneally by the bolus injections of 50 mg?kg -1 , 100 mg?kg -1 and 100 mg?kg -1 , respectively, immediately after the induction of SCI. And then the effects of Res were observed at 1 h, 24 h, 48 h after SCI compared with MPSS. RESULTS Res could obvious inhibit the secondary spinal cord edema with the most remarkable suppressing rate by 11 5% at 48 h. Res was able to significantly suppress the activities of the lactic dehydrogenase with the highest suppressing rate for over 40% at 24 h. In the meantime, Res markedly improved the Na +, K + ATPase activities that promoted to the biggest extent to 60% at 48 h. The effects of Res were the same as or even stronger than those of MPSS. CONCLUSION Res may effectively protect the spinal cord from secondary spinal cord edema and improve the energy metabolism system in SCI. It suggested that Res may be having potent therapeutic effect on SCI.

Transient expression of BMP12 gene transfer to mesenchymal stem cells / 解放军医学杂志

Objective To investigate the feasibility of the committed differentiation of mesenchymal stem cells(MSCs) into tenocytes,the present study was carried out. Method NMP12 gene transfer to MSCs was performed with pEGFP C1 expressing vector by electroporation. Results Green fluorescence protein(GFP) expression was detected as early as 6h postelectroporation, peaked at 12h and could maintain 3 days. And then, GFP expression of some cells was weakened. Conclusion The results of the present study indicated that the successful transfection of MSCs was made, an the committed differentiation of MSCs into tenocytes was practicable

THE BIOLOGICAL FEATURES OF RHESUS BONE MARROW MESENCHYMAL STEM CELLS / 解剖学报

Objective In order to explore the biological features of bone marrow mesenchymal stem cells. Methods The ALPase activity, Vimentin and Laminin were detected by immunocytochemistry. Cell cycle and surface antigenic features were analyzed by flow cytometry technique. Cell growth curve was measured by MTT test. The telomerase activity was detected by in situ hybridization. Results The ALPase activity and Laminin were negative, Vimentin was positive in the MSCs. The percentage of G1, S, and G2 of cell cycle was 89.5%,7.8%,7.7%, respectively. MSCs were positive for CD166、CD29、CD44、CD105, and negative for CD34 and HLA-DR. The average doubling time of cells was 32 hours. The telomerase activity was positive. Conclusion The cultured cells were not hemopoietic stem cells or fibroblasts, but the MSCs that had not differentiated. Furthermore, it had relatively high proliferation ability.

MITOCHONDRIAL CHANGES AND THEIR REGULATION INMURINE APOPTOTIC PERITONEAL MACROPHAGE / 解剖学报

Objective To study the changes in activity of NADPH oxidase, the effects of signal molecules on membrane potential and ROS production of mitochondria in apoptotic murine peritoneal macrophages. Methods Laser scanning confocal microscopy, flow cytometry and fluorescence labeling were used. Results 1 The macrophages treated with dexamethasone developed apoptosis quickly and presented concomitant apoptotic changes. 2 Mitochondria membrane depolarized quickly, the activity of NADPH oxidase declined sharply, and ROS production decreased rapidly. The erasers of ROS promoted macrophage apoptosis. 3 PKC favored, and cAMP inhibited the macrophage apoptosis and the rapid drop in ROS and mitochondrial membrane depolarization. cGMP and TPK which slightly inhibited macrophage apoptosis, had no effects on ROS. Conclusion 1 The activity of NADPH oxidase declined sharply, hence the ROS decreased rapidly, which promoted apoptosis in macrophages treated with dexamethasone. 2 The signal molecules affected apoptosis by modulating ROS decline and mitochondria depolarization. The results suggested that, mitochondria variations, especially the variations of ROS and membrane potential, mainly affected macrophage apoptosis.;