BACKGROUND: The increasing prevalence of diabetes mellitus has been become one of the diseases which threaten the heath of human being in the 21st century. Islet transplantation is considered to be the most effective approach to cure type Ⅰ diabetes mellitus. However, lack of donor tissue limits the application of this therapy. However, recent progress of stem cell research shows that stem cell therapy may be a potential means to solve this problem.OBJECTIVE: To take activin A and all-trans retinoic acid (AR) in inducing the differentiation of bone marrow mesenchymal stem cells (MSCs) and explore its possibility DESIGN: A randomized controlled experiment.SETTING: Institute of Biotechnology, Academy of Military Medical SciencesMATERIALS: This experiment was conducted at the Institute of Biotechnology, Academy of Military Medical Sciences from November 2004to June 2005. Six male Sprague-Dawley rats, with body mass of 150-160g, were provided by the Experimental Animal Center of Academy of Military Medical Sciences.METHODS: Femoral bone marrow of the rats was extracted under aseptic condition. Bone marrow mesenchymal stem cells (MSCs) were isolated with density gradient centrifugation. Passaged MSCs were randomly divided into 4 groups: high concentration of glucose (HG), AR, beta-mercaptoethanol (ME) and negative control groups. MSCs were induced to differentiate into IPCs with conditional medium containing high concentration glucose, activin A, RA and ME etc. After induction, phenotypes of differentiated cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: Expression of insulin and glucagon of differentiated cells were examined by immunocytochemistry. Insulin-1 mR-NA expression of differentiated cells was detected by RT-PCR.RESULTS: After bone marrow mesenchymal stem cells were induced,there were scattered insulin-and glucagon-positive cells in the HG group,many insulin-and glucagon-positive cells in the AR and ME groups, and these cells formed insulin-like structure. The expression of insulin-1mRNA could be observed in the HG, AR and ME groups. Insulin-and glucagonpositive cells and the expression of insulin-1mRNA were not observed in the negative control group.CONCLUSION: We adopt an induction scheme based on AR and other matured factors, and successfully make bone marrow mesench.ymal stem cells induce and differentiate into insulin positive reaction cells and form insulin-like structure, but its induction efficiency needs further improvement.

Objective To investigate the prognosis of local resection in patients with low rectal cancer, and assess surgical indications for this procedure. Methods One hundred and twenty-four patients with low rectal cancer from Jan 1975 to Dec 2006 were analyzed, the clinicopathologic features and surgical, outcome were examined as prognostic factors. Survival rate was estimated by Kaplan-Meier method and compared by Log-Rank test, prognostic factors were analyzed by multivariate COX proportional hazards model. Results The 5-year survival rate of 124 patients underwent local resection was 90.7 %(97/107), there were 4.8 %(6/124) patients with complications and 15.3 %(19/24) ones with local recurrence.The infiltration, vascular invasion, the size of tumor and the histological grade were significant prognostic factors of overall survival, but gender, age, the tumor site and the macroscopic type were not. Multivariate analysis indicated that the tumor infiltration were independent poor prognostic factor. Conclusion Local resection is suitable for Tis and T1 low rectal cancer, and those with high local recurrence factors should undergo radical resection. Strict follow-up and adjuvant therapy is necessary for local excision.

OBJECTIVETo investigate the clinical effect of lateral superior genicular composite tissue flap for tissue defect.

METHODSThe axis line of flap is the lateral thigh vertical midline. The cutaneous branch is inserted 4 cm near the femoral lateral epicondylus. The anterior border is the elongation line along patellar lateral border. The posterior margin is the hinder margin of femoral biceps. The lower border is the horizontal line along the upper line of patella. The composite flaps were used in 18 cases with soft tissue defects in extremities, 11 cases with clacaneus tenden defects and 16 cases with bony nonunion. Results From Mar. 2002 to Sept. 2013, 45 cases were treated with the composite tissue flaps. The flaps size ranged from 6 cm x 3 cm to 17cm x 9 cm. All the flaps survived completely. Blood supply crisis happened in 2 cases, which was released by reanastomosis. The patients were followed up for 1 - 2. 5 years with satisfactory aesthetic and functional results. All the bone defect and nonunion were healed. Good healing was also achieved in donor sites. 8 months after operation, knee joint function is evaluated as good by hospital special surgery knee score (HSS).

CONCLUSIONLateral superior genicular compostie tissue flap can be used to reconstruct soft tissue defect, bone defect and tenden calcaneus defect in one stage.

Anatomic Landmarks ; anatomy & histology ; Follow-Up Studies ; Graft Survival ; Humans ; Knee ; anatomy & histology ; Muscle, Skeletal ; anatomy & histology ; Soft Tissue Injuries ; pathology ; surgery ; Surgical Flaps ; transplantation ; Thigh ; Time Factors ; Wound Healing

OBJECTIVETo investigate the therapeutic effect of primary reconstruction of skin avulsion injury with bilateral anterolateral thigh flaps combined with thorax umbilicus flap or latissimus dorsi flap.

METHODSFrom June 2005 to Aug. 2011, 4 cases with skin avulsion injury on both feet were treated. The bilateral anterolateral thigh flaps, including with anterolateral thigh cutaneous nerves, were transferred to cover the feet plantar. The thorax umbilicus flap or latissimus dorsi flap were used to cover the feet dorsum.

RESULTSAll the skin avulsion injury were reconstructed primarily. All the flaps survived completely with good cosmetic and functional results. The patients were followed up for 6 months to 2 years with good sensory recovery (two point discrimination: 14-18 mm).

CONCLUSIONThe skin avulsion injury on both feet can be primarily reconstructed by bilateral anterolateral thigh flaps combined with thorax umbilicus flap or latissimus dorsi flap.

Adolescent ; Follow-Up Studies ; Foot Injuries ; surgery ; Humans ; Lacerations ; surgery ; Myocutaneous Flap ; transplantation ; Reconstructive Surgical Procedures ; Skin ; injuries ; innervation ; Surgical Flaps ; innervation ; transplantation ; Thigh ; innervation

Objective To investigate the feasibility of laparoscopic radical resection for colorectal cancer in patients with respiratory dysfunction. Methods A total of 64 patients with colorectal cancer with respiratory dysfunction simultaneously admitted in our hospital. Following the principles of en-bloc resection, thirty-six patients underwent laparoscopic radical resection, and 28 underwent open resection. Results The time of postoperative oxygen inhalation was shorter in laparoscopic group than that in open resection group (3.5 d vs 4.6 d) (P<0.05), and independently expectorating was better in laparoscopic group than that jn open resection group (P <0.05). The time to endotracheal intubation removal (21.2 min vs 23.9 min) and oxygen saturation were no significant difference between laparoscopic group and open resection group. One case got lung infection in open resection group, both groups had no atelectasis, respiratory failure and urinary infections case. Conclusion Laparoscopic surgery is feasible for colorectal cancer in patients with respiratory dysfunction.

Objectives To approach the method and clinical effect on tendo calcaneus and complex tissue defect with microsurgery repair.Methods Retrospective summary the methods of 356 cases with tendo calcaneus and complex tissueserious defect,which repaired by different microsurgery from June 1994 to March 201 1.Two type were divided on account of different degree of serious tendo calcaneus and complex tissue defect.Type A:the length of tendo calcaneus defect was less than 3 cm,and cutaneous deficiency is less than 3 cm × 20 cm.Direct suture (166 cases) or Abraham retrograde V-Y method (72 cases)was used to repair endo calcaneus defec,anfregional flap metastasis was used to repair cutaneous deficiency.Two hundred and thirtyeight cases were used by those methods,including of lateral heel flap repair(23 cases),medial plantar island flap(58 cases),instep island flap(40 cases),above medial malleolus flap(48 cases),above ateral malleolus flap (24 cases),sural nerve nutrient vessel flap (29 cases) gastrocnemius muscle flap (16 cases).Type B:the length of tendo calcaneus defect was more than 3 cm,and cutaneous deficiency was more than 3 cm × 20 cm.Direct suture could not repair tendo calcaneus,the complex tissue flap free grafting was used to primary repair tendo calcaneus and complex tissue defect.One hundred and erghteen cases were used by those methods,including of tensor fasciae latae flap free grafting (52 cases),lateral above knee complex tissue flap free grafting (26 cases),latissimus dorsi muscle fascia flap free grafting (24 cases),rectus abdominis muscle front sheath flap free grafting (16 cases).Three hundred and fifty-six cases were repaired by these methods,including 238 cases of regional flap transfer 118 cases of tissue flap free grafting.Results In 238 cases of regional flap transfer,two hundred and twenty-six cases were successful,and 12 cases were partly success,which were wound healing by change dressings.In 118 cases of tissue flap free grafting,one hundred and nine cases were successful,and blood vesse articulo were happened to 8 casess,which were success by operations research,and 1 case was failure which had to use another tissue flap.Follow-up visit was dane from 1.0 year to 4.5 years after operation (average 3.2 years).Functional assessment according to the Thermann ralted the results as excellent in 240 csaes,good in 86 cases,common in 22 cases and worse in 8 cases.The fineness rate was 91.6％.Conclusion Microsurgical repair is a good method to tendo calcaneus and complex tissue defect,different method and strategy selected actively by tissue defect degree of tendo calcaneus and complex tissue can achieve satisfactory functional rehabilitation purpose.

Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.
Animals ; CHO Cells ; Cricetinae ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Protein C ; biosynthesis ; genetics ; RNA Interference ; Recombinant Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics ; metabolism ; Transfection

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.
Animals ; Batch Cell Culture Techniques ; CHO Cells ; Cell Cycle Proteins ; genetics ; Cell Line ; Cricetinae ; Cyclin-Dependent Kinase 2 ; genetics ; Cyclin-Dependent Kinase 6 ; genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Smad4 Protein ; genetics ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Genetic Engineering ; Insulin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; metabolism