Objective To construct small RNAi plasmid vectors which can suppress the expression of TITIN in Wistar rat. Methods A recombinant plasmid vector involving shRNA, which matched the base pair of rat TITIN N2B region mRNA perfectly, was constructed and transfected into normal neonatal cardiomyocytes and mesenchymal stem cells (MSCs) induced by 5-aza cytidine respectively. The expression of TITIN Z band was investigated by immunofluorescence. Results The expression of TITIN was weakened after recombinant plasmid was transfected into neonatal cardiomyocytes and MSCs induced by 5-aza cytidine. Conclusion The recombinant plasmid was constructed successfully, and it actually can block the expression of TITIN.

BACKGROUND:Both calcium phosphate cement II and recombinant human bone morphogenetic protein have certain osteoinductive effects, which have the possibility of repairing tendon-bone interface injury. OBJECTIVE: To investigate the osteoinductive effect of calcium phosphate cement II and its biomechanics analysis of repairing tendon-bone interface injury. METHODS:Five out of 35 adult healthy New Zealand white rabbits were randomly selected and their bilateral shoulder joint tendon-bone interface specimens were taken as normal control group after being sacrificed. The remaining 30 rabbits were used to make animal models of tendon-bone interface injury and then randomly divided into experimental and model groups. Rabbits in the model group had no treatment, and those in the experimental group were treated with calcium phosphate cement II. RESULTS AND CONCLUSION: After repair with calcium phosphate cement II, the injured tendon-bone interface of rabbits was obviously restored, and the repair effect became better with time. The expression level of bone morphogenetic protein 2 was also increased accordingly. The maximum tensile strength and the maximum stiffness of the injured tendon-bone interface were obviously increased. These results demonstrate that calcium phosphate cement II combined with recombinant human bone morphogenetic protein has good osteoinductive and repair effect in repair of tendon-bone interface injury.

Objective To establish a system for detecting integration frequency of antibiotic resist-ante integron.Methods We cloned integron and aadA2 gene cassette into different sites of plasmid pACYC 184,and the plasmid was transformed into E.coli BL21(DE3)containing plasmid overexpressing integrase.The positive clone was cultured overnight and then was spread on LB agar plate with or without streptomycin respectively,and with appropriate amount of bacteria.Clones after cultured overnight were counted to detect the integration frequency.Meanwhile we used positive clones in LB agar plate containing streptomycin as templates to carry out PCR.The purified PCR products were sequenced to identify the integration sites.Re-suits The integration frequency of integron capturing aadA2 gene cassette in BL21(DE3) host was 1.1 x 10-3 mainly at attI site.Conclusion This system can be used to detect integration frequency.

OBJECTIVE: To explore the effect of artesunate (ART) on cell differentiation and cell cycle distribution of the prostate cancer cell line PC-3 in vitro. METHODS: PC-3 cells were cultivated with ART from logarithmic growth phase. After 48-hour treatment, the cell cycles were detected by flow cytometry (FCM), and enzyme linked immunosorbent assay was used to detect the level of prostate specific antigen (PSA) in cell culture supernatant. The change of cellular morphology was observed under a transmission electron microscope (TEM). RESULTS: In comparison with the blank control group, the rate of G(0)/G(1) plus S stages of PC-3 cells was significantly decreased in the high-dose ART group. The PC-3 cell was arrested in G(2)/M by ART. The rates of G(2)/M of the high-dose ART group and the medium-dose ART group were obviously higher than those of the blank control group and the cisplatin group (P<0.05). The levels of PSA in the three ART groups were significantly lower than that of the normal control group (P<0.05, P<0.01). In the ART groups, TEM showed that some vacuoles appeared in endochylema, cell polarity was enhanced, cell nucleus leaned to one side of the cell, and microvilli increased on the other side of the cell. CONCLUSION: ART can induce PC-3 cell cycle arrest and differentiation in vitro.

BACKGROUND:Inducing factor and chondrogenic microenvironment is a primary factor, which influences chondrogenic differentiation and chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). OBJECTIVE:To explore the feasibility of in vivo chondrogenesis by co-culture of bone marrow-derived MSCs and chondrocytes. DESIGN, TIME AND SETTING:A randomized controlled animal experiment was performed at Department of Pathology, Stomatological Hospital, Fourth Military Medical University of Chinese PLA between September 2004 and March 2005. MATERIALS:Fifteen New Zealand rabbits of clean grade were used for cell-scaffold construct transplantation. The rabbits were randomly divided into co-culture, chondrocyte, and bone marrow-derived MSC groups, with 5 rabbits in each group. Five neonatal New Zealand rabbits, aged 1-3 days, were used for isolation and culture of bone marrow-derived MSCs and chondrocytes. Polyglycolic acid (PGA) scaffold material (Shanghai Yikuo Company, China) has a fiber diameter of 15 μm, with an average interval of 150-200 μm, an interval porosity of 97% and 2-mm thickness. METHODS:In the co-culture group, bone marrow-derived MSCs and chondrocytes were mixed at a ratio of 3:1. The mixed cells were seeded onto a pre-wetted PGA scaffold (5 mm×5 mm )at the ultimate concentration of 6.0×1010 L-1. Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum was dropwise added to peripheral compound for 1 week of culture. In the chondrocyte, and bone marrow-derived MSC groups, chondrocytes and bone marrow-derived MSCs of the same ultimate concentration were seeded respectively onto the PGA scaffold. Then, the cell-scaffold constructs were transplanted into subcutaneous tissue of adult rabbits. MAIN OUTCOME MEASURES:Gross observation and hematoxylin-eosin & Masson staining of neo-cartilage were performed after in vivo culture for 8 weeks. RESULTS:Cell in all groups had a fine adhesion to the scaffold. In both co-culture and chondrocyte groups, the cell-scaffold constructs could maintain the original size and shape during in vivo culture and formed homogenous mature cartilage after 8 weeks of in vivo culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features. In the bone marrow-derived MSCs group, connective tissue rather than cartilage was found during in vivo culture. CONCLUSION:Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of bone marrow-derived MSCs and thus promote the chondrogenesis of bone marrow-derived MSCs in vivo.

Objective To report the clinical observation and nursing points for recurrent or refractory and aggressive non-Hodgkin's lymphoma treated with MINE regimen. Methods Patients (46 cases) with recurrent or refractory and aggressive non-Hodgkin's lymphoma were given mental nursing,preparation and evaluation of the condition of patients before chemotherapy. Complications especially extravasation of infusion fluid should be prevented during chemotherapy and myelosuppression and long-term complications should be prevented after treatment. Results No patient died after treatment but several complications such as infection(14 cases), nausea and vomiting (grade Ⅰ～Ⅱ, 22 cases, grade Ⅲ,2 cases), alopecia (grade Ⅰ～Ⅱ, 27 cases), mucositis (grade Ⅰ～Ⅱ, 4 cases), liver function lesion (grade Ⅱ, 1 case) were found and improved after proper treatment. No nephrotoxicity and hemorrhagic cystitis were found. Conclusions Selective mental nursing,proper chemotherapy preparation,evaluation of patients' condidtion and positively prevention of chemotherapy-related complications could increase the chemotherapy effect of patients with recurrent or refractory and aggressive non-Hodgkin's lymphoma.

Objective To investigate the relationship between acute biliary pancreatitis (ABP) and anomalous pancreaticobiliary duetal union (APBDU). Methods 131 patients with ABP were enrolled to test the serum total bilirubin (TB), alanine amintransferase (ALT), aspartate amintransferase (AST), alkaline phosphates (ALP), γ-glutamyl transferase (γ-GT). All the patients received medical treatment, and then these tests were performed again. Thereafter, all the patients underwent selective surgery and intra-operative cholangiography was performed to observe the pancreaticobiliary duetal union. Results 27 patients (20.6%) with APBDU were found in 131 patients. Among them, 8 cases (29.6%) was B-P subtype (TypeⅠ), 16 cases (59.3%) was P-B subtype (TypeⅡ) , and the remaining 3 cases was mixed subtype (TypeⅢ). A significant decrease of ALT, AST, ALP, γ-GT after non-surgical treatment in both group of APBDU and NAPBDU was noted (P＜0.05). The serum levels of ALT, AST,γ-GT in APBDU patients were (71.81± 23.19) U/L, (47.85±27.87) U/L, (52.86±31.49) U/L, respectively; and in NAPBDU patients were (51.96±15.40) U/L, (40.77±16.58) U/L, (34.86±26.47) U/L. The difference was statistically significant (P＜0.05). Condusions APBDU is an important etiology of ABP.

Objective To observe the prokinetic effect of domperidone on colon and compare with that of mosapride and cisapride.Methods ① In viva experiment:forty rats were divided into control,domperidone,mosapride,and cisapride groups with 10 in each group.Stran gauges were planted both in proximal and distal colons and colonic motor activities were recoded in conscious rats.② In vitro experiment:the prokinetics effects of dopamine or dopamine combined with domperidone on the contraction of isolated rat colon strips were recorded by tone-transducers in the thermostatic muscle bath.Results In viva experiments:① in the interdigestive period of resting,activities of rhythimic phasic contraction were recoded in colon at conscious rats,and ② it had been showed that dornperidone significantly enhanced the contraction of colon in dose-dependent manner.Compared with control group,the mean contraction amplitude of proximal colon and distal colon increased by 84.61% ± 7.26% and 76.37 % ± 8.47% in domeridone group,respectively,which was higher than those in mosapride group (50.32%±8.16% and 45.13%±7.16%,respectively),but as same as those in cisapride group (92.55% ± 8.37% and 81.27% ± 9.95%,respectively).In vitro experiments:① perfusion of dopamin (40 mg/ml) could significantly decrease the contraction of isolated colon strip by 91.56% ± 10.24% in comparison with Krebs-Ringer solution,and ① domperidone could block the relaxant effect of dopamin on the isolated colon strip.Conclusions Domperidone can significantly enhance the colonic motor activities via antidopaminergic action.The effects of domperidone are similar as eisapride and greater than mosapride.

Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .

Objective To screen out specific microsatellite markers for use in Tupaia belangeri chinensis genetic testing. Methods Firstly to screen about 700 microsatellite loci from whole genome.Secondly to choose about 100 better loci without defect factors.Lastly 46 primers were designed by 33 tree shrew’ s microsatellite loci obtained from whole genome and other references.Agarose gel electrophoresis and polyacrylamide gel electrophoresis were used for PCR products, and better loci based on electrophoresis results were chosen.Then STR scan was used to select the microsatellite loci combination for genetic testing.Results Twenty-two microsatellite loci were selected with a significant Stutter peak on STR scanning.Comparing the alternative loci and ultimately selected loci, there were two loci available in the five alternative loci of T.glis.The coincidence rate between T.glis and T.b.chinensis was 40%.There were two loci available in the five alternative loci of T.minor, and the coincidence rate between T.minor and T.b.chinensis was 40%.There were two loci available in the three alternative loci of T.belangeri, and the coincidence rate between T.belangeri and T.b. chinensis was about 70%.Conclusions The 22 microsatellite loci screened in this study are well applied for genetic testing of Tupaia belangeri chinensis, therefore, provide a scientific basis for the genetic quality monitoring of tree shrews.