BACKGROUND: Because of the non-homology of protein and gene between human and animals, to promote osteogenesis or spinal fusion of animals by construction of tissue-engineered bone with the human gene has influenced the experimental validation.OBJECTIVE: To construct the seed cell line for bone tissue engineering with stable expression of human bone morphogenetic protein 2 (hBMP2).METHODS: The full-length hBMP2 gene was cloned from human muscle tissues by nested RT-PCR and transfected to human bone marrow mesenchymal stem cells (hBMSCs) with lipidosome. The transfected hBMSCs were cultured with G418 in vitro to screen and purify the cells. A series of analyses such as RT-PCR, dot-ELISA, immunohistochemstry and alkaline phosphatase activity analysis were performed to evaluate the situation of hBMP2 expression and secretion at 48 hours and 3 weeks after the transduction. hBMSCs transduced with empty plasmid and the normal hBMSCs served as positive control and blank control groups, respectively, which were used for observation of cell growth, proliferation and biological characteristics of transfected cells. RESULTS AND CONCLUSION: The transfected hBMSCs appeared in small groups or clusters, and had a good proliferation after subculture in vitro. Some G418-resistance cell clones and calcium nodules were found when cultured with G418 in vitro. No significant difference was noted in the cell proliferation between the hBMP2 transfection group and two control groups. The ALP activity in the hBMP2 transfection group remained significantly higher than that in the two control groups (P < 0.01). At 48 hours and 3 weeks after transduction, hBMSCs could express actively hBMP2 by RT-PCR monitoring, and had a positive reaction of dot-ELISA and immunohistochemical analysis. The expression of hBMP2 gene in the experiment group at 48 hours was significantly higher than that at 3 weeks after transduction while there was no expression of hBMP2 gene in the two control groups. The above results show that the hBMSCs transfected by hBMP2 gene not only have potentials of normal proliferation and osteogenic differentiation, but also can stably express hBMP2.

BACKGROUND:In clinical application, the structure of crista lambdoidalis of L5 was unclear. It needs to expose more tissue to define L5 entry point through transverse process or superior and inferior articular process. This increased the risk of trauma and iatrogenic superior intervertebral degeneration. Therefore, it is necessary to expose L5 entry point with a minimaly invasive way. OBJECTIVE:To investigate the accuracy of L5 pedicle screw insertion with the entry point of mastoid process slope by imaging. METHODS:Mastoid process was located on the base of L5 superior articular process. A cant was formed when the highest point of L5 mastoid process backward protuberance extended inwards and downwards. The cant was defined as mastoid process slope; it was lateral to pedicle medial superior side internaly, medial to transverse process root and superior to the top of crista lambdoidalis. The slope was first easily touched and exposed in lumbar posterior surgery through paraspinal muscle space approach. Fifty patients of lumbar spine disorders were treated by L5 pedicle screws fixation through the entry point of mastoid process slope. According to preoperative radiographic and CT images, pedicle screw insertion direction of the sagittal and transverse sections was calculated. The diameter of pedicle screw was 6.5 mm. The condition of intraoperative successful rate of screws placement at one time was analyzed. The accuracy of screw placement was evaluated by postoperative radiographic and CT images. RESULTS AND CONCLUSION:With the method of the mastoid process slope, the successful rate of screw placement at one time was 96% (96/100). Totaly 100 screws were inserted into L5. According to the criterion by Gertzbein, 95 screws (95%) totaly located in pedicles and 5 screws (5%) encroached on the pedicle from medial wal. Three (3%) out of 5 inaccurately placed screws cut out less than 2 mm of the inner wal, while 2 (2%) between 2 mm and 4 mm, without neurologic deficits. The method of mastoid process slope had a high successful rate of screw placement. Combined with preoperative X-ray films and CT images could obtain a high accuracy rate of screw insertion.

OBJECTIVE： To study the improvement effect and mechanism of methylated urolithin A on oleic acid-induced lipid accumulation in human liver cancer Huh-7 cells. METHODS： Oleic acid was adopted to induce lipid accumulation model cells. Huh-7 cells were divided into control group （culture medium）， model group （1 mmol/L oleic acid）， low-dose group （1 mmol/L oleic acid+10 μmol/L methylated urolithin A） and high-dose group （1 mmol/L oleic acid+20 μmol/L methylated urolithin A）. Oil red O staining was used to observe lipid accumulation in cells. Triglyceride（TG） enzyme assay was applied to determine the TG content in cells. PCR was employed to detect the mRNA expression of FASN， SREBP-1， PPAR-α and PPAR-γ in cells. Western blotting was used to determine the protein expression of FASN in cells. RESULTS： After induced by oleic acid， a large amount of lipid droplet accumulated around the cells； the intracellular lipid and TG content， mRNA expression levels of FASN， SREBP-1 and PPAR-γ， protein expression levels of FASN were increased significantly， while mRNA expression level of PPAR-α was decreased significantly （P＜0.01）. After intervened with methylated urolithin A， lipid droplet around the cells decreased significantly； the contents of lipid and TG in cells were decreased significantly， while the mRNA expression levels of FASN， SREBP-1 and PPARγ and protein expression level of FASN were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Methylated urolithin A can improve oleic acid-induced lipid accumulation in Huh-7 cells， the mechanism of which may be associated with inhibiting fat synthesis， promoting lipid metabolism and down-regulating the expression of metabolism-related factors as FASN， SREBP-1 and PPAR-γ.