N-Alkylation of Amines with Alcohols Catalyzed by Manganese(II) Chloride or Bromopentacarbonylmanganese(I).

A manganese-catalyzed N-alkylation reaction of amines with alcohols via hydrogen autotransfer strategy has been demonstrated. The developed practical catalytic system including an inexpensive, nontoxic, commercially available MnCl2 or MnBr(CO)5 as the metal salt and triphenylphosphine as a ligand provides access to diverse aromatic, heteroaromatic, and aliphatic secondary amines in moderate-to-high yields. In addition, this operationally simple protocol is scalable to the gram level and suitable for synthesizing heterocycles such as indole and resveratrol-derived amines known to be active for Alzheimer's disease.

[Chondrogenesis of bone marrow mesenchymal stem cells induced by transforming growth factor beta3 gene in Diannan small-ear pigs].

OBJECTIVE: To observe transforming growth factor beta3 (TGF-beta3) gene expression and the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) after TGF-beta3 gene is transfected into BMSCs of Diannan small-ear pig. METHODS: Recombinant adenovirus 5 (rAd5) was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-beta3, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs (weighing, 12-15 kg), and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAd5-TGF-beta3 as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-beta3 protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot. RESULTS: The rAd5-TGF-beta3 recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours (transfection efficiency of 84.86%). Immunofluorescence staining showed that the expression of TGF-beta3 protein was obvious at 72 hours; Western blot showed that there was a TGF-beta3 positive band with a relative molecular mass of 30 x 10(3), while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positive band with a relative molecular mass of 130 x 10(3) at 21 days after culture, while the control group and blank control group had no positive band. CONCLUSION: rAd5-TGF-beta3 gene can be successfully transfected into BMSCs via adenovirus vectors, and stable expression of TGF-beta3 protein can be observed, enhancing BMSCs differentiation into chondrocytes, which may provide an experimental basis for gene therapy of joint cartilage defects.

[CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR ß3 GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS].

OBJECTIVE: To construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2 (BMP-2) and transforming growth factor ß3 (TGF-ß3) genes, to observe the expressions of BMP-2 and TGF-ß3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs. METHODS: BMP-2 cDNA and TGF-ß3 cDNA were amplified by PCR, and were subcloned into the pEC3.1 (+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-ß3 plasmid respectively. They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-ß3. The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured. The 3rd passage BMSCs were transfered with Ad-BMP-2 (group A), Ad-TGF-ß3 (group B), Ad-BMP-2+ Ad-TGF-ß3 (group C), and untransfected cells served as a control (group D). The expressions of BMP-2 and TGF-ß3 genes and proteins were detected by PCR, immunofluorescence, and Western blot. The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type II. RESULTS: The Ad-BMP-2 and Ad-TGF-ß3 were constructed successfully and confirmed by PCR and sequencing. The expression clones of Ad-BMP-2 and Ad-TGF-ß3 were packaged into maturated adenovirus successfully, the titer was 5.6 x 10(8) and 1.6 x 10(8) pfu/mL respectively. The PCR results showed a light band at 310 bp in group A and at 114 bp in group B, and both 310 bp and 114 bp bands in group C, but no band in group D. The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B, respectively; in group C, both red and green fluorescence expressions were detected, and no red or green fluorescence was detected in group D. The results of Western blot showed that there was a light band at 18 x 10(3) in group A and at 50 x 10(3) in group B; both 18 x 10(3) and 50 x 10(3) bands were detected in group C; but no band was detected in group D. The cells were positive for collagen type II in groups A, B, and C; group C acquired strong collagen type II staining when compared with group A and group B; in group D, the cells were negative for collagen type II staining. CONCLUSION: The recombinant adenovirus vector expressing BMP-2 and TGF-ß3 are constructed successfully. The BMP-2 and TGF-ß3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection, which could afford modified seeding cells for cartilage tissue engineering.

[Effectiveness of computer-assisted preoperative planning system in anterior cruciate ligament reconstruction].

OBJECTIVE: To evaluate the feasibility and effectiveness of computer-assisted preoperative planning system-ACL Detector in anterior cruciate ligament (ACL) reconstruction. METHODS: Between March 2009 and January 2012, 80 patients with ACL rupture received arthroscopic ACL single-bundle reconstruction with autologous hamstring tendon transplantation. Before operation, the preoperative planning was done by computer-assisted preoperative planning system-ACL Detector (trial group, n=40) or by conventional method (control group, n=40). There was no significant difference in gender, age, disease duration, injury cause, preoperative Lysholm score, and preoperative International Knee Documentation Committee (IKDC) score between 2 groups (P > 0.05). After operation, the effectiveness was evaluated by Lachman test, pivot shift test, Lysholm score, and IKDC score; the digital three-dimensinal model of knee was reconstructed, and the impingement rate of ACL graft was measured. RESULTS: All incisions healed by first intention, and no complication was found. The patients were followed up 18-25 months (mean, 20 months) in trial group and 18-24 months (mean, 21 months) in control group. The Lysholm score and IKDC score were significantly increased at 18 months after operation when compared with preoperative scores (P < 0.05), but no significant difference was found between 2 groups (P > 0.05). The results of Lachman test and pivot shift test at 18 months after operation were significantly better than those before operation in 2 groups (P < 0.05), but no significant difference between 2 groups after operation (P > 0.05). MRI showed that impingement was observed in 1 case of trial group (2.50%) and in 8 cases of control group (20.00%), showing significant difference (chi2=4.51, P=0.03). CONCLUSION: The computer-assisted preoperative planning system-ACL Detector could be successfully applied to ACL reconstruction. It has the same improvement in knee functional score as conventional surgery, but it is better than conventional surgery in reducing the impingement incidence.

Relationship between DNA ploidy,expression of ki-67 antigen and gastric cancer metastasis.

AIM:To evaluate the relationship between the expression of Ki-67 antigen and the pathobiological behaviours of gastric cancers especially their distant metastases.METHODS:Fifty-six specimens of gastric cancer routinely fixed in formalin and embedded in paraffin (FFEP) were studied by immunohistochemical method.RESULTS: Expression of Ki-67 antigen was significantly related to the distant metastases to liver, ovary and adrenal gland (P < 0.01), but not related to the histological type, growth pattern, depth of invasion, histological differentiation and the metastases to local lymph nodes (P > 0.05).Furthermore, the Ki-67 antigen expression was significantly related to the DNA aneuploidy pattern, which is closely related to poor prognosis (P < 0.05).CONCLUSION:Overexpression of Ki-67 can be used as an objectiv marker of the proliferative activity for predicting prognosis of gastric cancer and metastatic potential to distant organs.