Objective To analyze and identify the mutations of ATP6V0A4 and ATP6V1B1 gene in autosomal recessive distal renal tubular acidosis (rdRTA) children,and study the association of genotype and phenotype. Methods Genome DNA was amplified by PCR.Mutations of ATP6V0A4 and ATP6V1B1 gene in 3 children from 3 families were examined by direct sequencing.One hundred unrelated healthy subjects were selected to evaluate all mutations found in this study. Results A novel homozygous nonsense mutation was identified in ATP6VOA4 gene in one child, and a novel heterozygous nonsense variant and a frame-shift alteration were found in another child.No mutation of both genes was found in the third child.Conclusions Study of mutant genes of rdRTA in Chinese patients is helpful to understand the association in genotype and phenotype and increase the level of cognition and treatment to this disease.

BACKGROUND: Cyclooxygenase 2, aggrecanase 1, and insulin-like growth factor 1 are involved in pathological injury of the articular cartilage. OBJECTIVE:To observe the expression of shRNA vectors carrying cyclooxygenase 2, aggrecanase 1 and overexpression vectors carrying insulin-like growth factor 1 in bone marrow mesenchymal stem cels. METHODS:Lentiviral vectors carrying the silencing gene cyclooxygenase 2, aggrecanase 1, the over-expressing gene insulin-like growth factor 1 and binding green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect passage 3 human bone marrow mesenchymal stem cels culturedin vitro (experimental group). The human bone marrow mesenchymal stem cels transfected with no target gene lentivirals were used as negative control group. The human bone marrow mesenchymal stem cels transfected with no treatment served as blank group. RESULTS AND CONCLUSION:Cyclooxygenase 2 and aggrecanase 1 transfected in human bone marrow mesenchymal stem cels were significantly inhibited at gene and protein levels, while the expression of insulin-like growth factor 1 was increased significantly at gene and protein levels. We confirmed that cyclooxygenase 2 and aggrecanase 1 were successfuly silenced while insulin-like growth factor 1 overexpressed by using lentiviral vectors in human bone marrow mesenchymal stem cels, which brings a new hope for the systemic gene treatment of arthritis.

Objective:To observe the effects of hydrogen on osteogenic capacity of human periodontal ligament cells(hPDLCs)stim-ulated with P.g-LPS.Methods:hPDLCs were cultured and divided into 4 groups:control(C)group,osteogenic induction(OI) group,OI +1 00 ng/ml LPS(OILPS)group and OIPLS +3%H2 (H2 OIPLS)group,and treated respectively.Alizarin red staining (ARS)was carried out 3 weeks after treatment.ALP and OC mRNA expression of the cells was examined by RT-PCR after 7-d treat-ment.Results:LPS decreased A value of ARS(P <0.01 ),ALP mRNA expression(P <0.001 )and OC mRNA expression(P <0.001 )of the cells.H2 increased the A value(P <0.05),ALP mRNA expression(P <0.01 )and OC mRNA(P <0.01 )of the cells treated by LPS.Conclusion:High concentration of P.g-LPS can inhibit osteogenic capacity of hPDLCs,while hydrogen can impair the P.g-LPS induced suppression of hPDLC's osteogenesis.

BACKGROUND: Bone morphogenetic protein 2 and transforming growth factor β are important factors in bone regeneration, increasing the expressions of bone morphogenetic protein 2 and transforming growth factor β can promote the osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To construct the lentivirus vector carrying bone morphogenetic protein 2 and transforming growth factor β3, and to observe the expression of lentivirus vector in bone marrow mesenchymal stem cells. METHODS: The recombinant lentiviral vectors carrying transforming growth factor β3, bone morphogenetic protein 2 and green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect the passage 3 rabbit bone marrow mesenchymal stem cells in vitro cultured (transfection group). The bone marrow mesenchymal stem cells transfected with single gene lentivirals (single gene transfection group) carrying transforming growth factor β3 and bone morphogenetic protein 2 or single lentivirals were as control (control group). At 1 week after trasfection, the total RNA and protein were extracted from each group for detection. RESULTS AND CONCLUSION: The green fluorescence bone marrow mesenchymal stem cells transfected with transforming growth factor β3 and bone morphogenetic protein 2 gene for 3 days could be observed under fluorescence microscope, and the transfection efficiency was over 90%. Reverse transcription-PCR and Western blot results showed the mRNA and protein expressions of transforming growth factor β3 and bone morphogenetic protein 2 in the transfection group were higher than those in the single gene transfection group and the control group. The results indicate that lentivirus can successful y transfect transforming growth factor β3 and bone morphogenetic protein 2 into the bone marrow msenchymal stem cells and achieve its high expression, and these two genes have the synergistic effect of promoting expression.

Objective To construct bone marrow stem cell sheets and to investigate its effects in the process of osteogenesis.Methods BMSCs were differentiated into osteoblasts and then seeded into a temperature responsive culture dish to construct BMSC sheets.PLGA scaffolds in which both BMSC suspension and BMSC sheets were added,were implanted into the left side of the dogs' mandible.In the other side,PLGA scaffolds that were not wapped with BMSC sheets were implanted as control.At 16 weeks,the samples were processed for radiological analysis and histological examination.Results Cells in the BMSC sheets grew well.In the experimental side,the optical density of the samples was higher than that of the control side (P＜0.05) and plenty of lamellar bones and Haversian system were observed.Conclusions The formation of lamellar bones can be promoted by PLGA scaffolds and BMSC sheets in the process of tissue engineering bone reconstrution.

Objective:To construct recombinant lentiviral vectors harboring interference RNA ( RNAi ) targetting murine TNF-αgene,so as to lay the foundation on the RNAi gene therapy.Methods: Three small interfering RNA ( siRNA) sequences targeting murine TNF-αgene ( siRNA1,siRNA2,siRNA3) and negative-control siRNA were designed and synthesized.The inhibition effects of siRNAs on TNF-α,IL-1βand IL-6 secretion of LPS-stimulated RAW264.7 macrophages were observed using real-time PCR and ELISA methods.DNA oligo was designed and synthesized according to the most effective siRNA 2 sequence.The recombinant lentiviral shuttle plasmid expressing short hairpin RNA ( shRNA) was constructed and sequenced.The lentiviral shuttle plasmids with packaging plasmids were transfected into 293T cells to produce lentiviral particles.Results: ①The TNF-αmRNA relative expression levels of siRNA1, siRNA2 and siRNA3 were 0.24±0.01,0.16±0.02,0.19±0.01 respectively,significantly lower than that of negative control (0.95± 0.02) (F=531.3,P<0.001).The inhibition rates at mRNA level were 74.26%,83.09%,79.93%,respectively comparing with negative control.No significance was observed in IL-1βor IL-6 mRNA relative expression change after TNF-αsiRNA transfection ( P>0.05).②The TNF-αprotein expression levels of siRNA1,siRNA2 and siRNA3 were (23.95±1.21),(17.27±1.46),(19.07± 1.57)ng/ml respectively,significantly lower than that of negative control (35.37±2.93)ng/ml (F=18.1,P=0.000 6<0.001).The inhibition rates of protein expression were 32.29%, 51.16%, 46.08%, respectively comparing with negative control.③The PCR product electrophoresis showed that recombinant vectors yielded 343 bp fragments,non-constructed vectors yielded 306 bp fragments.DNA sequencing partially showed insertion sequence.④Lentiviral particles were obtained by transfecting 293T cells with recombinant lentiviral shuttle plasmids and lentiviral packaging plasmids.Cells grew well during virus production with strong fluorescence expression.The titer of concentrated virus was 2×106 TU/μl.Conclusion:The lentiviral vector harboring RNAi targeting murine TNF-αgene has been successfully constructed.

BACKGROUND:Using experimental animals to simulate diseases of human being is the basis of studying etiology and treatment of the diseases, so the diseases of nasal cavity and sinus need suitable experimental animals as models.
OBJECTIVE:To observe the regional anatomy of rhino-sinus in rabbits and its performance through CT imaging, and to discuss the feasibility of applying a rabbit model to the study of animal rhino-sinusitis.
METHODS:Routine coronal and axial scanning images of rhino-sinus of New Zealand rabbits were performed through Discovery CT750 HD. The rhino-sinus anatomy was then observed.
RESULTS AND CONCLUSION:The nasal septum is located on both sides of the nasal cavity. The lateral wal of rabbit nasal is composed of maxil ary turbinate, middle turbinate, the inside of the middle turbinate and inferior turbinate. The maxil ary sinus cavity is the largest one and ethmoid sinus, sphenoid sinus and frontal sinus are relatively much smal er. Al these sinuses are paired and symmetrical. The rhino-sinus in rabbit is displayed clearly in CT scan. The anatomical location of rabbit is similar to that of human;however, the maxil ary sinus of rabbit is greater than that of human correspondingly, which is suitable for operating and applying to surgical anatomy and imaging analysis. The rabbit model of rhino-sinus can be applied to simulate human rhino-sinusitis.

Objective To investigate the correlation of the expressions of angiopoietin-2 (Ang-2) and angiopoietin-2 receptor(Tie-2)in serum and placenta with preeclampsia. Methods From May 2009 to April 2010, 62 women with preeclampsia who delivered in Affiliated Hospital of Qingdao University Medical College were recruited in the study, including 30 women with moderate preeclampsia (MPE group) and 32 women with severe preeclampsia (SPE group). Another 30 healthy pregnant women were taken as control group. ELISA was used to measure the serum Ang-2 in these women. Semiquantitative reverse transcription (RT)-PCR was used to investigate the expressions of Ang-2 mRNA and Tie-2 mRNA in placenta. Western blot was used to determine the expression of Ang-2 protein in placenta. Results (1) The serum concentrations of Ang-2 in MPE group and SPE group were (5.4 ± 1.8) μg/L and (5. 1 ± 1.7) μg/L,respectively. Both were significantly lower than that in control group (16. 2 ± 4. 5) μg/L (P<0. 01).There was no significant difference between MPE group and SPE group (P > 0. 05). (2) The expressions of Ang-2 mRNA in placenta of MPE group (2. 1 ± 0. 7) and SPE group (2. 0 ± 0. 6) were both significantly lower than that of control group (5.8 ± 0. 8; P<0. 01). But there was no significant difference in Ang-2 mRNA expression between MPE group and SPE group (P>0. 05). (3) No significant difference was found in the expressions of Tie-2 mRNA in placenta among MPE group (1. 33 ±0. 04), SPE group (1.35 ±0. 05) and control group (1.34 ± 0. 04; P > 0. 05). (4) The expressions of Ang-2 protein in placenta of MPE group (2.0 ± 0. 8) and SPE group (2. 0 ± 0. 8) were both significantly lower than that of control group (5.7 ±0. 9; P <0. 0l), while no significant difference was found between MPE group and SPE group (P >0. 05) . (5) In MPE group and SPE group, the serum concentrations of Ang-2 were positively correlated with the levels of Ang-2 mRNA and Ang-2 protein in placenta(r =0. 651, 0. 627; P <0. 01). Conclusions Decreased expressions of Ang-2 mRNA and Ang-2 protein in placenta reduced serum concentration of Ang-2. Low expression of Ang-2 may be involved in the pathophysiological process of preeclampsia by affecting the formation of placenta in early pregnancy.

Objective To investigate the effects of lentiviral-mediated RNA interference (RNAi) targeting tumor necrosis factor-α(TNF)-αgene on the expression of TNF-α, interleukin (IL)-1β, IL-6 of murine macrophages RAW264.7, and the efficiency of RNAi experimental gene therapy for the murine collagen-induced arthritis (CIA). Methods The RAW264.7 macrophages were infected by lentivirus-RNAi particles, then stimulated by Lipopolysaccharides (LPS). The TNF-α, IL-1β, IL-6 expression of RAW264.7 macrophages were measured with real-time polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA). CIA models were esta-blished in DBA/1 mice using bovine type Ⅱ collagen. The treatment effect of lentivirus-RNAi on CIA were observed through arthritis scores, serum TNF-α measurement and hind paw paraffin section hematoxylin/eosin staining after lentivirus-RNAi particles tail vein injection. Results The TNF-αmRNA relative expression level of lentiviral RNAi group was 0.291 ±0.021, significantly lower than that of negative control group 0.925±0.013 (t=25.4, P<0.01). The inhibition rate in mRNA levels was 68.5%. The serum TNF-α level of lentiviral RNAi group was [(249 ±11) ng/ml], significantly lower than that of negative control [(382±6) ng/ml] (t=10.31, P<0.05). The inhibition rate of protein levels was 34.7%. It had no effect on the IL-1β and IL-6 mRNA expression. On the 8th day after systemic administration, the arthritis score of lentivirus-RNAi group was 2.50±0.19, which was significantly lower than that of blank controls (3.63 ±0.18) and negative controls (3.75 ±0.16) (F=42.8, P<0.01). From now on, arthritis score of lentivirus-RNAi group and positive control decreased slowly to at least 2 weeks after treatment induction. The serum TNF-α levels of lentivirus-RNAi group and positive controls were [(35±6) pg/ml] and [(32±7) pg/ml] significantly lower than that of negative controls [(47±3) pg/ml] (t=3.03, 4.11, P<0.01) respectively. Morphological examination showed that the lentivirus-RNAi decreased CIA pathological manifestations. Conclusion Lentiviral-mediated RNAi targeting murine TNF-α gene can effectively inhibit TNF-α expression both in vitro and in vivo, which also effectively improve the CIA arthritis score. Lentiviral-mediated RNAi targeting TNF-αgene provides a potential strategy for rheumatoid arthritis (RA) treatment.

BACKGROUND: As the preliminary experiment for gene therapy in intervertebral disc degeneration, this study aims to construct a recombinant plasmid containing fluorescent pAAV-hSOX9-IRES-tdTomato for adeno-associated virus packaging, in a broader attempt to lay the foundation for late experiments in vitro and in vivo.OBJECTIVE: To construct human SOX9 gene overexpressing adeno-associated virus, pAAV-hSOX9-IRES-tdTomato, packaging. METHODS: The plasmid pAAV-IRES-tdTomato and plasmid pUC57-hSOX9 were connected into pAAV-hSOX9-IRES-tdTomato by enzyme digestion method. The adeno-associated virus was packaged with plasmid co-transfections method. The recombinant pAAV-hSOX9-IRES-tdTomato was transfected into 293AAV cell by calcium phosphate transfection. The purification and drop of adeno-associated virus was tested by determination of biological titer. RESULTS AND CONCLUSION: The results of BLAST sequence comparison analysis showed that, pAAV-hSOX9-IRES-tdTomato exactly matched the synthetic gene sequence hSOX9. The titer is 1×107 TU/mL. Human gene SOX9 recombinant adenoviruses, pAAV-hSOX9-IRES-tdTomato, have been constructed successfully.