Objective To establish a quantitative detection method for Mycobacterium tuberculosis by immunomagnetic capture combined with PCR-ELISA detection system with double internal standards(IMC-PCR-ELISA) .Methods The immunomagnetic (Dynabeads? ) which could specifically capture Mycobacterium tuberculosis were prepared .According to Mtp40 and IS6110 gene sequence of Mycobacterium Tuberculosis ,2 pairs of primers(upstream primer was modified with Biotin at 5′end) ,2 same-length mutant fragments with PCR amplified fragments ,and 3 capture probes(modified with digoxigenin at 3′end) were designed .Myco-bacterium tuberculosis were captured by immunomagnetic ,then detected by PCR-ELISA with double internal standards .Results The IMC-PCR-ELISA method could yield quantitative results in about 4 h with a detection limit at 5 × 103 copies/mL .There was a fine linear relationship between the copies of Mtp40(IS6110)in fact and in the calculation through formula when the concentrations of low internal standards were 30-70 copies/mL and the concentrations of high internal standards were 8 000-12 000 copies/mL (r2 =0 .998) .No nonspecific amplification was observed .Conclusion A rapid and quantitative method for the detection of Myco-bacterium tuberculosis was established successfully .The IMC-PCR-ELISA method was rapid ,sensitive ,secific and quantitative .

Objective To observe the effect of the method of warming kidney and dredging collaterals on the clinical effect and the content of urine C5b-9 in patients with idiopathic membranous nephropathy with spleen kidney yang deficiency and blood stasis. Methods A total of 60 idiopathic membranous nephropathy patients with spleen kidney yang deficiency and blood stasis type were randomly divided into the conventional western medicine treatment group (control group), Jingui-Shenqi pill and Taohong-Siwu decoction plus conventional western medicine treatment group (treatment group), 30 cases in each group. The Scr was detected by deproteinized alkaline picric acid method, and BUN was detected by rate method, and serum albumin (ALb) was detected by bromocresol green dye binding method, and 24 hours urinary protein was measured by pyrogallol red colorimetry, and the double antibody sandwich ELISA method was used for detection of urinary C5b-9. Results The Jingui-Shenqi pill combined with Taohong-Siwu decoction plus conventional western medicine treatment has obvious curative effect on patients. The total effective rate was 83.3 in the treatment group (25/30), and the control group was 60％ (18/30). After treatment, the Alb (33.5 ± 7.95 g/L vs. 28.8 ± 6.10 g/L, t=2.569) in the treatment groupwas significantly higher than that in the conventional treatment group (P<0.01). While the 24 h urine protein (2.40 ± 0.92 g/24 h vs.3.60 ± 2.3 g/24 h, t=2.653), the contents of C5b-9 in urine(42.5 ± 17.50 ng/mg vs.71 ± 25.2 ng/mg, t=5.088) in the treatment group were significantly lower than those in the conventional treatment group (P<0.01). Conclusions The method of warming kidney and dredging collaterals can improve the clinical symptoms, improve serum albumin level, reduce the 24 hour urine protein and urinary C5b-9 content of idiopathic membranous nephropathy of spleen and kidney yang deficiency and blood stasis type.

Objective To explore the molecular mechanism of Yishen Huoxue prescription in delaying the development of renal fibrosis by regulating the microRNA-126/vascular endothelial growth factor-Notch (miR-126/VEGF-Notch) signaling pathway. Methods Ninety male Sprague-Dawley (SD) rats were randomly divided into sham operation group (sham group), unilateral ureteral obstruction (UUO) model group, losartan group (50 mg·kg-1·d-1) and high, medium and low doses Yishen Huoxue prescription group (25.2, 12.6, 6.3 mL/kg). Each treatment group began to administer drugs by gavage on the day when UUO modeling was finished until the end of the experiment. Left renal tissues of rats were harvested after 7, 14 and 21 days postoperatively. The pathological changes of renal tissue were observed after hematoxylin and eosin (HE) and Masson staining under the microscope, and the renal fibrosis score was calculated. The mRNA expressions of renal tissues miR-126, VEGFA, vascular endothelial growth factor receptor-2 (VEGFR-2), Notch1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results① Pathology results: the kidney tissue of sham group was normal. In UUO model group, renal tubules expanded and inflammatory cells in renal interstitium increased; renal tubulointerstitial fibrosis could be seen 7 days after operation, and the degree of fibrosis was gradually increased with time. The renal fibrosis score at each time point after operation in UUO model group was significantly higher than that in sham group. Compared with UUO model group, each treatment group were improved the degree of renal swelling and atrophy of renal parenchyma; the score of renal fibrosis were significantly decreased in the middle and high doses Yishen Huoxue prescription group and losartan group at the 7th day after operation (1.00±1.00, 0.91±0.58, 1.01±0.58 vs. 2.00±0.00,all P < 0.01); but there was no significant difference between low dose Yishen Huoxue prescription group and UUO model group. ② RT-PCR results: Compared with sham group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissues were significantly increased at each time point after operation in UUO model group. Compared with the UUO model group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissue of 7 days postoperatively in the middle and high doses Yishen Huoxue prescription group and the losartan group were significantly increased [miR-126(2-ΔΔCt):0.465±0.067, 0.639±0.092, 0.404±0.069 vs. 0.132±0.021; VEGFA(2-ΔΔCt): 0.024±0.005, 0.027±0.007, 0.023±0.006 vs. 0.014±0.006; VEGFR-2(2-ΔΔCt):0.021±0.007, 0.023±0.008, 0.019±0.007 vs. 0.012±0.004; Notch1(2-ΔΔCt):0.017±0.004, 0.020±0.005, 0.018±0.005 vs. 0.007±0.004; all P < 0.05]; compared with the losartan group, the mRNA expressions of each index in the middle and high doses Yishen Huoxue prescription group were increased at all the time points, but there was no significant difference between them (all P > 0.05). There was no significant difference in mRNA expression of each index between low dose Yishen Huoxue prescription group and UUO model group. Conclusions The medium and high doses of Yishen Huoxue prescription can effectively antagonize renal fibrosis. Yishen Huoxue prescription may use up-regulation the signaling pathways of miR-126/VEGF-Notch to mediate renal microvascular newly born, eventually to improve renal microvascular damage and delay the development of renal fibrosis progression.