Objective To evaluate the influence of tissue factor pathway inhibitor 2(TFPI-2)gene on the proliferation and invasion of K562.Methods The expression vector pcDNA3.0/TFPI-2 was transfected into human leukemia line K562 cells(K562-T)by using liposome,then the mRNA and protein TFPI-2 were detected by real-time RT-PCR and western blot separately.The growth curve and the colony-forming unit assay were used to measure the ability of cells growth and transwell chamber model was employed to test the ability of cell invasion in vitro.Results Expression of mRNA and protein of TFPI-2 was detected in transfected cells.The growth rate and self-replication ability of K562-T cells were lower than those of the two control groups obviously.The number of K562-T cells to traverse a Matrigel-coated membrane was dramatically decreased compared with that of non-expressing cells.Conclusion The gene of TFPI-2 can inhibit the growth,proliferation and invasion of the K562 cells.

Objective To investigate sequence variations of 12 miRNA genes in multiple myeloma(MM) in order to find whether sequence variations in miRNA genes are associated with tumorigenesis and discuss the clinical significance of MM associated with miRNA genes mutations. Methods The miRNA gene mutations in 20 cases of MM, 4 MM-derived cell lines and 20 controls were detected by the methods of polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) and silver staining technique. Both clinical features and laboratory results were analyzed simultaneously. Results The electrophoretic patterns showed a total of three variations in miR-19a, miR-19b and miRNA-335,which were observed in 3 MM cells (15 %, 3/20). We also found variations of miRNA-335 in MM-derived cell lines KM-3and RPMI8226. However, no sequence alteration in the miRNA genes was observed in our set of controls. One of the three MM patients died, and two of them were detected mutations at the terminal stage of the disease.Conclusion A relative high frequency of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. And, detecting miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease.

Objective To construct a recombinant adenovirus vector of Hairpin RNA specific for MRP1 gene and study its inhibition of MRP1 gene expression in K562/AS2 cell line resistant to AS_2O_3 (ATO). Methods A MRP1-specific hairpin RNA recombinant adenovirus vector was constructed and used to infected K562/AS2 cells. Expression level of MRP1 mRNA detected by real-time fluorescent quantitative PCR. MRP1 protein detected by flow cytometry. MTT method was used to detected the cytotoxicity of ATO and etoposide. Results MRP1 mRNA and protein expression level in K562/AS2 cells before and after the pAd-MRPl-shRNA adenovirus infection was (34.70±0.28 vs 4.19±0.03, P <0.05) and (26.40±0.16 vs 10.85±0.37, P<0.05), respectively. RR of K562/AS2 to arsenic trioxide and etoposide was (11.4078±0.3183 fold vs 1.6126±0.3015 fold, P<0.05) and (5.9141 ±0.0149 fold vs 1.7664±0.1038 fold, P <0.05), respectively. The reversal fold of ATO and etoposide was (7.2409±1.3668) and (3.3555±0.1886), respectively. Conclusion Successfully constructed pAd-EGFP-U6-shRNA-MRPl adenovirus vector, the vector of infection K562/SA2 cells can inhibit MRP1 gene expression and reverse the resistance of the ATO and etoposide.

Objective To investigate the reversal effect of Topo Ⅱα-shRNA and Topo Ⅱβ-shRNA on Topo Ⅱ gene in K562/AS2 cells. Methods Three pieces of Topo Ⅱα-shRNA and three pieces of Topo Ⅱβ-shRNA were designed,synthesized and transfected into K562/AS2 cells by liposome. Expression level of Topo Ⅱα and Topo Ⅱβ mRNA were determined by real time fluorescence quantitative PCR. The expressions of Topo Ⅱα and Topo Ⅱβ protein were assayed with flow cytometer. Results After treated with Topo Ⅱα-shRNA or Topo Ⅱβ-shRN A for 24 hours,the expression level of Topo Ⅱα mRNA and Topo Ⅱβ mRNA protein in K562/AS2 cells decreased at most (78.22±0.01) %,(31.17±1.27) % (P <0.05),and (57.36±0.01)%,(23.98±1.22) % (P <0.05) respectively. Conclusion The expression of Topo Ⅱ gene can be down-regulated after infected with Topo Ⅱ-shRNA in K562/ AS2 cell line.

ObjectiveTo investigate the gene expressions of TAZ and Wnt/β-catenin on the postosteogenic cells of mesenchymal stem cells(MSC)in multiple myeloma(MM)patients and to explore the potential therapeutic target of multiple myeloma bone disease (MBD).MethodsBone marrow mononuclear cells MNC from MM and controls were isolated,cultured,expanded and then induced to osteogenic differentiation.Realtime quantitative RT-PCR was employed to detect the osteogenic markers (TAZ,Wnt/β-catenin,OPN,OC,ALP and Cbf α1); and alizarin red staining for mineral deposition.The mRNA expressions of TAZ and Wnt/β-catenin in the two groups were analysed.ResultsAlizarin red staining was positive and the red calcium nodules were appeared on the post-osteogenic cells of MSC.The mRNA expressions of OC,ALP and Cbf α1 were 2.0958±0.5665,2.6670±0.3847,0.8463±0.3473,respectively,on the post-osteogenic cells of MSC in the experimental groups,which were significantly higher than those of pre-osteogenic cells(1.3487±0.9291,1.1452±0.6054,0.4439±0.2945) (t =2.171,6.709,2.795; all P ＜ 0.05).The mRNA expressions of OPN,OC,ALP and Cbf α1 were 2.1096±0.8267,2.8991±0.3531,4.3045±0.2844,1.3273±0.4075,respectively,on the post-osteogenic cells of MSC in the controls,which were significantly higher than those of pre-osteogenic cells (1.2200±0.9091,0.8780±0.3927,1.9161±0.2684,0.6736±0.2513) (t =2.289,12.103,25.134,4.411; all P ＜ 0.05).The mRNA expressions of OPN,OC,ALP,Cbf α1 were 1.2710±0.5636,2.0958±0.5665,2.6670± 0.3847,0.8463+0.3473,respectively,on the post-osteogenic cells of MSC in the experimental groups,which were significantly lower than those of control groups(2.1096 ±0.8267,2.8991 ±0.3531,4.3045±0.2844,1.3273±0.4075) (t =-2.650,-3.805,-10.822,-2.841; all P ＜ 0.05).The mRNA expression of TAZ and β-catenin were 2.2315±1.0723 and 0.5801±0.2159 on the post-osteogenic cells of MSC in MM patients,which were significantly lower than those of control groups (4.4140±0.8325,0.9516±0.2920) (t =±5.085,-3.235;both P ＜ 0.05).ConclusionThe gene expressions of OPN,OC,ALP and Cbf α1,the osteogenesis related genes,are increased in post-osteogenic cells of MSC,which showed the MSC have been successfully induced to osteoblasts.Comparing with control groups,the osteogenic potential of MSC in MM patients is lower.Based on the above research,TAZ and Wnt/β-catenin may present a novel target for the future therapy of MBD.

Objective To study the COLIXA3 gene polymorphism of patients with fluorosis and to explore the pathogenesis of COLIXA3 gene in endemic fluorosis.Methods Fifty one cases of patients with drinking-water borne fluorosis were selected as the case group in Xinzhou city,Shanxi province and 28 cases of healthy people were as the control group.Dental fluorosis was detected by Dean method and skeletal fluorosis was examined by X-ray.COLIXA3 of exon 5 gene product of 103 points was amplified by PCR and the gene locus genotype was sequenced.Results Ten cases of mild dental fluorosis,14 cases of moderate dental fluorosis,15 cases of severe dental fluorosis were detected among the 51 patients.The control group was free of dental fluorosis.All the 51 cases of patients with fluorosis had varying degrees of skeletal fluorosis,mainly osteosclerosis lesions,accounting for 86.27％(44/51 ),and mild skeletal fluorosis patients were all osteosclerosis lesions,and osteosclerosis lesions and multiple skeletal lesions were found among moderate and severe skeletal fluorosis patients in the case group,while control group had no skeletal fluorosis.The differences between genotypes of frequency distribution of AA,Aa,aa of COLIXA3 of case and control groups were not statistically significant [96.08％(49/51 ),3.92％(2/51 ),0.00％(0/51) and 96.43％(27/28),3.57％(1/28),0.00％(0/28),x2 =0.94,P ＞ 0.05].ConclusionsCOLIXA3 gene polymorphism is not significantly correlated to fluorosis.

Objective To explore whether different degrees of fluorosis influence the expression of cartilage COLIXA3 protein in fluorosis model rats. Methods Forty male Wistar rats 3 to 4 weeks old were randomly divided into 5 groups according to body mass, and these rats were fed with distilled water containing sodium fluoride(NaF) of 0(control), 25, 50, 100 and 150 mg/L for 6 months, respectively, in order to establish the animal model of drinking water type fluorosis. Pathomorphologieal changes of the osseous tissues of rats were analyzed under light microscope and transmission electron microscope, and the expression of COLIXA3 protein of femur metaphysis was examined by immunohistochemistry. Results HE staining showed different degrees of femoral metaphyseal ossification of cartilage in each experimental group, bone density increased, with sclerotic lesions of skeletal fluorosis. The control group showed no abnormal cartilage. Electron microscopy showed that the experimental groups with varying degrees of cartilage cell swelling, cell matrix fades, 50 mg/L group .showed hyperplasia, and 100,150 mg/L groups were observed with organelles decreased, part of the disintegration of the cartilage cell lacunae, lmmunohistochemical staining of rat chondrocytes COLIXA3 was positive, cytoplasm with brown granules, cartilage COLIXA3 protein expression(23.3 ± 4.5, 41.2 ± 5.6, 26.4 ~ 7.5) in the 25, 50 and 100 mg/L groups enhanced. Compared to the control group (6.1 ± 3.5), the expression of 50 and 100 mg/L groups was significantly increased, and the differences were statistically significant(all P < 0.05). The expression(13.3 ± 4.2)of COLIXA3 protein in 150 mg/L group was decreased compared with the previous three, but is still higher than that of control, and the difference was not statistically significant(P > 0.05). Conclusions There has pathological changes of sclerosing skeletal fluorosis in animal model. Low-dose fluoride promotes while high-dose inhibits cartilage cell proliferation. When fluorine concentration in external environment is too high and with extended exposure to fluoride, direct toxic effects of fluoride on cartilage cells is observed. Fluorine affects and promotes the expression of COLIXA3 protein in cartilage. Low-dose fluoride can promote COLIXA3 protein expression, as the dose increases (over 100 mg/L), the effect decreases.

ObjectiveTo study the relationship between expression of a3 chain of collagen Ⅸ (COLIXA3)mRNA in the population exposed to fluorine and fluorosis,in order to reveal the role of COLIXA3 gene in the pathogenesis of endemic fluorosis.MethodsTwelve cases of mild drinking water-born skeletal fluorosis were selected as case groups in Regiment 123 and 128 of Xinjiang Production and Construction Corps Seven Division,6cases of healthy people living in fluorosis areas for more than 10 years as a internal control group and 6 heathly cases living in non-fluorosis areas for more than 10 years as a external control group.The expression of COLIXA3mRNA of peripheral blood lymphocyte of skeletal fluorosis patients and control groups were determined by using SYBR Green Ⅰ chimeric fluorescent method for real-time quantitative PCR.ResultsThe results of the relative expression of COLIXA3 mRNA of case group,internal control group and external control group were 2.16 ± 0.62,1.06 ± 0.09 and 1.05 ± 0.12,respectively.The COLIXA3 expression in case group was significantly higher than that of the internal control group and the external control group (all P ＜ 0.05),while the difference of COLIXA3expression between the internal control group and the external control group was not significantly different (P ＞0.05).ConclusionsFluorine contributes to the expression of COLIXA3 mRNA in peripheral blood lymphocyte,and the expression is up to 2 times higher than that of the control groups,meaning potential biomarkers.

OBJECTIVETo investigate the vascular supply of intrinsic muscles of foot and anatomic basis for muscular flap design.

METHODSA radiopaque injectate (lead oxide-gelatin mixtures, 26 ml/kg) was injected into 10 fresh cadavers. The dissected regions were photographed and each intrinsic muscles on the foot was removed and radiographed. The number, type, diameter of vascular branches of muscles and their distributions were observed. The area of the vascular territory supplied by each source vessel was calculated using Scion Image for Windows software.

RESULTSThere were significant architectural differences among the intrinsic muscles. The muscles length varied from 22.5mm to 116.2mm [average, (66.1 +/- 23.2)mm]. The measured fiber length were relatively consistent, ranging from 14.2 mm to 27.5 mm [average, (20.2 +/- 4.5)mm]. There are 63 vascular branches into the 23 foot muscles, each muscle having average branches of 3.2 +/- 0.8. The average diameter of branches, the length and width of each vascular territorial area is (0.8 +/- 0.3) mm, (2.2 +/- 0.8) cm, and (0.9 +/- 0.4) cm, respectively. Other findings included that some muscles were not present in some cadavers.

CONCLUSIONSThe blood supply of intrinsic muscles of foot is abundant with different diameter and distributions of branches. There is an anatomic basis for muscular or musculoosseous flap design. There are 7 intrinsic muscles with large and reliable vascular supply which can be chosen as muscular flaps.

OBJECTIVETo observe the distribution of the blood vessels in the integument tissue of the channel area of legs.

METHODSThe integument tissue of the lower limbs in the 12 cadavers were dissected with macro-and micro-dissection, radiographical technique of systemic artery and technique of image pattern analysis to observe and analyze the origins, branches and anastomoses in the integument tissues along the channels of legs.

RESULTSThe distributional density of the blood vessels in the integument tissues of legs along the channel area of the three-yin meridians of the foot, the Gallbladder Meridian, and the Urinary Bladder Meridian was higher than that in the other parts. They formed an obvious nutrient vascular chain on the arteriogram. The distributional density in the channel area of the Stomach Meridian was not obviously increased and the obvious nutrient vascular chain could not be seen.

CONCLUSIONAn obvious nutrient vascular chain is formed in the integument tissue along the channel area of legs except the Stomach Meridian.