AIM: Utilizing mRNA fluorescent differential display RT-PCR in our previous study,we found that the mRNA expression level of an intermediate protein(IF)-desmuslin was dramatically down-regulated in the abnormal veins of patients with primary vein incompetence.In this study,we testified the alteration of desmuslin expressing style at gene transcription and translation levels.METHODS: Specific PCR primers were designed according to the sequence of desmuslin mRNA.The cDNA fragments of desmuslin obtained from differential display were labeled by DIG as specific probes.Then semi-quantitative RT-PCR and Northern blotting techniques were applied to investigate the expression level of desmuslin mRNA in normal and abnormal veins.Simultaneously,the specific single-cloned antibody bestowed by Yuji Mizuno was used to evaluate the amount of DMN protein in the two groups by Western blotting and immunohistochemical techniques.RESULTS: In the abnormal veins isolated from the patients with primary vein incompetence,the expression of desmuslin mRNA was significantly down-regulated,compared with that in control group(semi-quantitative RT-PCR: 0.19?0.05 vs 0.83?0.08,P

Objectives: To investigate the expression and distribution of survivin and P53 in human non small cell lung cancer (NSCLC),and to evaluate the relationship between survivin and P53. Methods:The expression of survivin and P53 in 95 patients with NSCLC were examined using immunohistochemical methods (SP), and the relationship between them and clinicopathological parameters was analyzed. Results: The positive rates of survivin and P53 in NSCLC were 62% and 55.8% respectively, in the normal lung tissues nearby were 17% and 7% respectively( P

Objective To assess the role of superficial vein and calf perforating vein in chronic venous insufficiency of lower limbs. Methods Consecutive seventy-five cases (78 limbs) of slight PDVI ( I? -Ⅱ?) were evaluated. Venous hemodynamics were detected by color duplex, color Doppler velocity profile technology and APG preoperatively, in the first month, the third month and the first year postoperatively. Data were statistically analyzed. Results Symptoms and signs disappeared in 88. 5% (69/78) limbs. The ulcer-healing rate was 92. 8% ( 13/14). The postoperative VFI at each check-point significantly decreased than that before operation (P0.01). Conclusions Superficial venous reflux can be effectively broken up by the operations. Venous stasis on calf effectively decreased by subfascial endoscopic perforator surgery (SEPS). But neither of the two procedures greatly improve the hemodynamic status of deep vein.

Objective: To investigate the expression and distribution of NOS2 and NOS3 in human non-small cell lung cancer (NSCLC),and to evaluate the relationship between their expression and tumor angiogenesis and lymph node metastasis. Methods: the expression of NOS2, NOS3 and IMVD in 95 patients with NSCLC were examined using immunohistochemical methods (S-P), and the relationship between them and many clinicopathological parameters was analyzed. Results: The positive expression of NOS3 was associated with histological subtype, IMVD and lymph node metastases of NSCLC(P

Objective To investigate the effect and mechanism of estrodial (E2) on intracellular free calcium in the endometrial-myometrial interface (EMI) smooth muscle cells from uteri with adenomyosis.MethodsFrom March 2011 to October 2011,16 uterus specimens were collected from patients with adenomyosis undergoing hysterectomy in Beijing Obstetrics and Gynecology Hospital,which included 9 proliferative endometrium and 7 secretory endometrium.EMI smooth muscle cells from the uterus were cultured and loaded with calcium ion ( Ca2 + ) fluorescent probe fluo-4/AM.The labeled cells were stimulated with the various concentration of E2 ( 1 × 102,1 × 103,1 × 104,1 × 105 pmol/L,respectively),then the changes of intracellular Ca2+ fluorescence intensity were measured by laser scanning microscopy.The most suitable concentration of E2 was selected,and the reaction difference between the EMI smooth muscle cells of two menstrual phases were also investigated; The changes of intracellular Ca2 + fluorescence intensity were detected proliferative and secretory smooth muscle cells in E2 conjugated to bovine serum albumin (17β-E2-BSA) group,cycloheximide (CHX) group,fulvestrant (ICI182780) group and pertussis toxin (PTX) group.Results ( 1 ) The cell viability of primary cultured EMI smooth muscle cells was well at 24 hours culture.(2) 1 × 102 - 1 × 105 pmol/L E2 can rapidly increase the intracellular Ca2+ fluorescence intensity within 1 min ( P ＜ 0.01 ) ;The increased amplitudes caused by 1 × 104 pmol/L and 1 × 105 pmoL/L E2 were the most significant,but there was no significant difference between them (P ＞0.05).1 × 104 pmol/L was the most suitable concentration.( 3 ) With the 1 × 104 pmol/L E2,the Ca2+ fluorescence intensity changes showed no significant difference between the EMI smooth muscle cells from the proliferative phase and secretory phase uterus (P ＞ 0.05 ).The Ca2+ fluorescence intensity changes were 646 ± 32 in 17β-E2-BSA group and 602 ±31 in CHX group,when compared with 513 ±26 and 617 ±35 in respective control group,no significant difference was observed (P ＞ 0.05 ).The increased amplitude of 188 ± 20 in the PTX group and 302 ± 11 in ICI182780 group exhibited significant difference with 632 ± 33 and 635 ± 24 in respective control group ( P ＜ 0.01 ).Conclusion E2 could increase the intracellular Ca2 + of EMI through a membrane receptor dependent and nongenomic mechanism of action.

Objective To investigate the regulation mechanism of estrogen on the free calcium of smooth muscle cells at the endometrial-myometrial interface (EMI) in uteri with adenomyosis. Methods From September 2011 to November 2012, 59 uterine myometrial specimens were obtained from 59 cases underwent hysterectomy, including 28 adenomyosis patients as adenomyosis (ADS) group and 31 patients with cervical intraepithelial neoplasia Ⅲ as control group. EMI smooth muscle cells were cultured and loaded with calcium ion fluorescent probe fluo-4/AM. After treated with trisphosphate (IP3) receptor antagonist, blocker of sarcoplasmic reticulum calcium-adenosine triphosphate (ATP), depleted agent of the ryanodine receptor-operated Ca2+, inhibitor of L-type calcium channel, inhibitor of Na+-Ca2+exchanger, the labeled cells were stimulated with estrogen. The changes of intracellular Ca2+fluorescence intensity were detected by laser scanning microscopy. The changes of intracellular Ca2+concentration was indicated byΔF[Ca2+]i. Results (1) Under normal calcium conditions, after the stimulation of estrogen, intracellular Ca2+fluorescence intensity in ADS group and control group both increased than those without estrogen. TheΔF[Ca2+]i in ADS group was 384±26, and in the control groupΔF[Ca2+]i was 235±20. TheΔF[Ca2+]i in ADS group was higher than that in the control (P<0.01). Without calcium conditions, theΔF[Ca2+]i in ADS group was 207 ± 17, and in the control group ΔF[Ca2+]i was 221 ± 19. TheΔF[Ca2+]i in ADS group was almost the same with the increase in the control (P=0.731). The ΔF[Ca2+]i in ADS group was significantly decreased compared with the calcium condition (P<0.01). However, there was no significant difference in the control between with and without calcium conditions (P=0.060). (2) After treated with IP3 receptor antagonist, blocker of sarcoplasmic reticulum calcium-ATP, depleted agent of the ryanodine receptor-operated Ca2+, theΔF[Ca2+]i in both groups were significantly reduced (P<0.05), the increase in ADS group was significantly higher than that in the control (P<0.05). (3)After treated with inhibitor of L-type calcium channel, theΔF[Ca2+]i in ADS group was 211 ± 19, while in the control group ΔF[Ca2+]i was 203 ± 16, and there was no significantly increased intracellular Ca2 +in both groups (P>0.05). But, the ΔF[Ca2 +]i in ADS group was significantly reduced after treatment compared to before treatment, (211 ± 19 vs 384 ± 28; P=0.001). The increase in control group was almost the same with before (203±16 vs 234±22, P=0.141). (4) After treated with inhibitor of Na+-Ca2+exchanger, theΔF[Ca2+]i in ADS group was 357 ± 24 and in the controlΔF[Ca2+]i was 209±19. The increase in ADS group was significant higher than that in the control (P=0.000). Compared withΔF[Ca2+]i on the condition without treating with inhibitor of Na+-Ca2+exchanger,ΔF[Ca2+]i was 363±21 in ADS group andΔF[Ca2+]i was 237±20 in control group after treatment. When compared with before treatment, there was no significant difference in both groups (P>0.05). Conclusions The increase of intracellular Ca2+induced by estrogen at EMI smooth muscle cells in adenomyosis patients was mostly from the release of arcoplasmic reticulum, and also from the Ca2+influx controlled by L-type calcium channel. The increase of Ca2+inducing abnormal contraction of EMI muscle may have relationship with the development of adenomyosis.

BACKGROUND: 6-hydroxydopamine, as an endogenous toxic factor in the pathogenesis of Parkinson's disease, participates in oxidative stress. N-acetylcysteine resists oxidation and removes free radicals effectively.OBJECTIVE: To investigate the toxicity of 6-hydroxydopamine in bone marrow stromal cells and the antagonistic effect of N-acetylcysteine on it. METHODS: Bone marrow stromal cells of Sprague-Dawley rats were cultured in vitro. Bone marrow stromal cells of passage 3 were treated with 6-hydroxydopamine with the final concentrations of 0,0.05,0.1g/L and N-acetylcysteine with the final concentrations of 0, 0.075,0.3,1.2,4.8g/L, respectively.RESULTS AND CONCLUSION: MTT assay showed that 6-hydroxydopamine (0.05 and 0.1 g/L) significantly decreased the viability of bone marrow stromal cells. This toxic effect of 6-hydroxydopamine was significantly inhibited by 0.3 g/L N-acetylcysteine. It suggests that antioxidant N-acetylcysteine may affect the toxic action of 6-hydroxydopamine.

Objective To evaluate clinical significance of two real-time fluorescence quantitative PCR kits for quantitative detection of HBV DNA and detection performance at different viral load levels.Methods A series of calibrators with different concentrations(1×106,5×105,1×105,5×104,1×104,5×103,1×103,5×102,1×102,1×101 kIU/L) were prepared with AB-type sera using the second generation WHO international standard (NIBSC code:97/750). HBV viral load in the sera of 78 patients,30 healthy blood donors and 10 calibrators were detected by real-time fluorescence quantitative PCR HBV DNA test kit from PIJI Bio-Technical Development Company Ltd (PG kit) and Cobas AmpliPrep/Cobas TaqMan HBV test kit. The correlation of the two methods was evaluated, and the performance of the two kits different viral load levels was evaluated. The false negative rate was analyzed. Negative control, low positive control and high positive control were included in every batch. Results Both two kits showed the correct results for the 10 specimens from the WHO international standards. The lowest detection limit of HBV DNA for Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit were 2.00 (kIU/L, lg) and 3.00 (kIU/L,lg) ,respectively. There was linear correlation between the results from Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit ( R2=0.938 7, P < 0.01 ), the upper limit of Roche kit had coincided with theoretical value. The samples with HBV DNA level above the upper limit of detection were diluted and retested to obtain the precise result. The result form Roche Cobas AmpliPrep/Cabas TaqMan HBV test [(8.35±0.20) kIU/L, lg] was higher than that from PG kit [(7.73±0.42 ) kIU/L, lg] (t=3. 776, P <0.05) . The detection of 108 serum samples showed that the level of HBV DNA detected by Roche Cobas AmpliPrep/Cobas TaqMan HBV test [(5.88±1.64) kIU/L, lg] was higher than that by PG kit [(5.25±1.55 kIU/L,lg] (t=12. 297 ,P <0.01 ). The correlation coefficients were high in samples with high HBV viral load[HBV DNA(>5.00 and≤7.00) kIU/L,Ig,R2=0. 779 7, P <0.01 ;HBV DNA( >7.00 ands≤9.00) kIU/L,lg,R2=0.603 7, P <0.01]. The correlation coefficient was low in samples with low HBV viral load[HBV DNA ( > 3.00 and≤5.00) kIU/L, lg, R2=0. 417 3, P <0.01 )]. When HBV DNA ( >3.00 and≤4.00) kIU/L,lg,the false negative rate was 33.3% (5/15). When HBV DNA ( > 1.08and≤3.00) kIU/L,lg,none of positive samples was detected with PG kit. Conclusions PG kit is not as good as Cobas AmpliPrep/Cobas TaqMan HBV test . The linear correlation between the results from the two kits is good. The correlation between the results detected with PG kit and Cobas AmpliPrep/Cobas TaqMan HBV test is higher in the high viral load groups than in the low viral load group. It is suggested that PG kit had a narrower linear range.

Objectives:To investigate the expression and distribution of b FGF in human non small cell lung cancer (NSCLC),and to evaluate the relationship between the expression of b FGF and the biological behavior of NSCLC, especially tumor angiogenesis and lymph node metastasis. Methods:The expression of b FGF and IMVD in 95 patients with NSCLC was examined using immunohistochemical methods (SP), and the relationship between the expression and many clinicopathological parameters were analyzed. Results: The positive rates of b FGF in carcinoma tissue (NSCLC) were much higher than those in the normal lung tissues nearby ( P 0.05). Conclusions: b FGF may play an important role in tumor angiogenesis and lymphatic metastasis of human NSCLC , and detection of b FGF may be a good metastasis and prognostic predictors for human NSCLC.

Objective:To investigate the effects of miR-125b on radiosensitivity of cervical cancer cells and its possible downstream mechanism.Methods:The expression of miR-125b and Foxp3 in cervical cancer tissues and cells was detected by RT-qPCR. The HeLa cells were irradiated with 0, 2, 4 and 6 Gy of X-rays. The expression of miR-125b and Foxp3 in each group was detected by RT-qPCR. After downregulation of miR-125b expression and 6 Gy X-ray irradiation, the proliferation ability of HeLa cells was detected by MTT assay, and the expression of Bax and Bcl-2 proteins were detected by Western blot. The relationship between miR-125b and Foxp3 was detected by Targetscan and Dual luciferin reporter assay. After downregulation of Foxp3 expression and 6 Gy X-ray irradiation, the proliferation ability of HeLa cells was detected by MTT assay, and the expression of Bax and Bcl-2 proteins were detected by Western blot. The effects of miR-125b on radiosensitivity of HeLa cells through Foxp3 were detected. After down-regulation of Foxp3, the contents of IL-10 and TGF-β in supernatant were detected by ELISA.Results:The expression of miR-125b in the tissues and cells of cervical cancer was significantly decreased, while the expression of Foxp3 was significantly increased. The expression of miR-125b in HeLa cells was increased after radiation in a dose dependent manner. The expression of Foxp3 in HeLa cells was decreased after radiation in a dose dependent manner. After 6 Gy X-ray irradiation of HeLa cells, down-regulation of miR-125b increased the cell proliferation capacity, significantly reduced the expression of Bax and increased the expression of Bcl-2. miR-125b targets Foxp3 and negatively regulates Foxp3 expression. After 6 Gy X-ray irradiation of HeLa cells, down-regulation of Foxp3 significantly reduced the proliferation capacity of HeLa cells, increased the expression of Bax and decreased the expression of Bcl-2. Overexpression of miR-125b can enhance radiosensitivity of HeLa cells through Foxp3.After 6 Gy X-ray irradiation, down-regulation of Foxp3 reduced the expression of IL-10 and TGF-β in cells.Conclusions:Upregulation of miR-125b enhances the radiosensitivity of cervical cancer cells by targeting and negatively regulating Foxp3, and the mechanism of that may be related to the down-regulation of Foxp3 to reduce the expression of IL-10 and TGF-β in the cells.