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

OBJECTIVE: To observe whether injection of medicine into low hydraulic resistance point along meridian brings about higher medicinal effect and to explore the efficacy of the theory that meridians are made up of channels featuring low hydraulic resistance by observing the diuretic effect of injecting furosemide or saline into the low hydraulic resistance point Shuifen (CV 9), vein and Zusanli (St 36) respectively. METHODS: Acute edema was induced in pigs by rapid intravenous injection of 2 000 ml normal saline. The pigs were divided into four groups: Shuifen (CV 9) injection of half dose furosemide group (SFF group), intravenous injection of full dose furosemide group (VF group), Zusanli (St 36) injection of full dose furosemide group (ZSLF group), and Shuifen (CV 9) injection of half dose normal saline group (SFS group). The accumulated urine quantity and the urine quantity generated in every 15-minute period were measured in each group respectively, every 15 minutes after injection, and the measurement lasted for two hours at one experiment. Each group involved eight times of experiments with one pig used for one experiment, which means the whole observation involved 32 times of experiments. RESULTS: The accumulated urine quantities observed in both SFF group and VF group were higher than those in the ZSLF group and the SFS group all through the measurement, showing significant differences during the period from the 15th minute to the 45th minute (P<0.05). But no significant difference was observed between the SFF group and the VF group during the whole 2-hour measurement (P>0.05). Analysis of urine quantity generated in every 15-minute period showed that diuretic effect climaxed during the 15th minute to the 30th minute in both SFF group and VF group. By contrast, ZSLF group reached diuresis climax during the 45th minute to 60th minute and no diuresis climax was observed in the SFS group all through the measurement. CONCLUSION: Injection of medicine into low hydraulic resistance point along meridian generates faster and more powerful medicinal potency, and this is likely to be applied to clinical practice. The theory that meridians are channels featuring low hydraulic resistance is important to the elucidation of meridians.

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.

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 improve the limitation and stability of tissue processing on mucosa from small intestine transplantation and to provide better evidence of pathological diagnosis for the acute rejection on small intestine transplantation. Method:92 samples of mucosa from small intestine transplantation were reviewed.There were three methods of tissue processing (ultrasonic wave and microwave as well as routine) were adopted.The results were analyzed with statistical methods. Results: Among 18 samples processed by the method of ultrasonic wave,4 samples were A grade (22.22%). Among 50 samples processed by the method of microwave,30 samples were A grade (60.00%). Among 24 samples processed by the method of routine,13 samples were A grade (54.17%).The Chi Square Test suggested that there was statistic difference among three processing methods. Conclusions: Microwave is the best method of tissue processing on mucosa of small intestine transplantation and for the diagnosis acute rejection.

Purpose To investigate the expression of EphA1 in renal cell carcinomas ( RCC) and to analyze its correlation with clini-copathological parameters in order to explore the clinical significance of EphA1 protein in renal cell carcinomas ( RCC) . Methods The immunocytochemical method was employed to measure the expression of EphA1 in 144 clear cell RCC ( ccRCC) tissues, 18 chro-mophobe RCC tissues and 6 papillary RCC tissues. Correlation between EphA1 protein expression and clinical parameters was evaluated by statistics. Results High level of the expression of EphA1 was observed in all normal renal tubes. The EphA1 protein was negative-ly or weakly expressed in 93 out of 144 ccRCC (64. 6%) and positively expressed in 51 out of 144 ccRCC (35. 4%). Positive expres-sion of EphA1 was observed in all samples of chromophobe RCC and papillary RCC. The high level expression of the EphA1 protein was significantly associated with younger patients (P<0. 001), sex (P=0. 016) and lower nuclear grade (P<0. 001). No significant relation between the expression of EphA1 and tumor diameter was found ( P=0. 316 ) . Conclusion EphA1 protein may be a new marker for the prognosis of ccRCC.

ObjectiveTo investigate the continuous pathological features of biopsy specimens from five cases of small bowel allotransplantation (SBT) in order to provide more reliable information for the diagnosis and treatment of acute rejection (AR) in SBT.Methods324 biopsy specimens of intestinal mucosa after SBT from 5 patients were collected and studied by histology,histochemistry and electron microscopy.ResultsIn the early stage after operation (0～3 months),AR IND-1 grade was diagnosed for four times on 3 of 5 patients.During 3-6 months,AR IND-1 grade for three times was diagnosed in 2 cases,and AR 2 grade for two times during 7 ～ 12 months. All the patients suffered ischemia reperfusion injury, lymphatic vessel reconstruction and AR.Conclusion The pathological examination of biopsy specimens of intestinal mucosa is still the most reliable detecting method to diagnose AR,and continuous observation may play an important role to monitor the occurrence,development,and treatment response of AR. The final diagnosis of AR depends on structure of intestinal mucosa,crypt epithelium injury and inflammatory cells infiltration. The communication among the pathologist and surgeon is the best way to reduce misdiagnoses.Ultrastructural examination is used to verify the pathogenic microorganism.

Objective To investigate the levels, phenotypes and functions of regulatory T cells (Treg) and Th17 cells in systemic sclerosis (SSc) patients and assess the role of imbalance between Treg and Th17 cells in the pathogenesis of SSc. Methods Peripheral blood mononuclear cells (PBMCs) of 31 SSc patients and 33 healthy controls were analyzed for the expression of CD4, CD25, CD45RA, the cytotoxic T-lymphocyte antigen-4 (CTLA-4), FoxP3, and IL-17 using flow cytometry. Treg immunosuppression capacities were measured in co-cultured experiments. The expressions of IL-17A, IL-22, IL-6 and IL-1β were measured by FlowcytoMix. The expression of FoxP3, CTLA-4, IL-17A, and RORC mRNA were measured by real-time polymerase chain reaction (PCR). The independent samples t-test was used to compare data between the groups. Results The frequency of Treg cells was significantly elevated in SSc [(3.6 ±1.1)%] compared with controls [(2.0±0.8)%, t=6.88, P<0.01)] and the expression of CTLA-4 was lower in Treg cells of SSc (P=0.034). The expression of CTLA-4 and FoxP3 mRNA was lower in SSc compared with controls (P<0.05), the immunosuppressive capacity of Treg cells were diminished in SSc (P=0.034). Th17 cells were increased in SSc (P<0.01); the levels of IL-17A, IL-22, IL-6 and IL-1β in the serums of SSc were higher than the controls (P<0.05). In SSc, CD25+FoxP3+IL-17+cells were increased[0.075%±0.032)%vs (0.049±0.027)%, t=3.52, P=0.029], but no difference of CD25+FoxP3+IL-17- was observed when compared with control. The expression of IL-17A and RORC mRNA were increased in SSc (P<0.01, respectively). Conclusion Both Treg and Th17 cells are increased in patients of SSc. Treg increases along with the deficiency of phenotype and function. FoxP3+IL-17+co-express cells are increased, and the balance between Treg and Th17 cells is broken.