BACKGROUND:The etiological factor for rheumatoid arthritis remains unclear, but the effects of nuclear factor-κB on the onset of rheumatoid arthritis have been gradual y paid great attention by rheumatologists.
OBJECTIVE:By using the RNA interference (RNAi) technique to block the signal pathway of nuclear factor-κB
mRNA of human rheumatoid arthritis synovial cells, this study explored its application prospect in the treatment of rheumatoid arthritis.
METHODS:The synovial cells were isolated, digested, and cultured for further use. In accordance with the
design principle of smal interfering RNA (siRNA), target sequences of siRNA of nuclear factor-κB were identified, and siRNA expression vector of nuclear factor-κB was synthesized and constructed. The four pGenesil-1/nuclear factor-κB siRNA expression vectors were transfected into the first passage of synovial cells that wel grew. Blank and negative control groups were set. cells at 12, 24, 48, 72 hours, 5 and 7 days after transfection were col ected, and RNA was extracted. Intracellular nuclear factor-κB mRNA expression levels were measured, and siRNA plasmid vector that could effectively inhibit nuclear factor-κB mRNA expression was screened out.
RESULTS AND CONCLUSION:Nuclear factor-κB highly expressed in synovial cells after human rheumatoid arthritis. 3#pGenesil-1/nuclear factor-κB apparently suppressed nuclear factor-κB mRNA expression in synovial cells with human rheumatoid arthritis. RNAi technique blocked nuclear factor-κB mRNA expression. Therefore, the block of nuclear factor-κB signal pathway might be a good target for rheumatoid arthritis gene therapy.

Objective To examine the expression of CD8+CD28- T cells in the peripheral blood and explore their roles in the pathogenesis of AS. Methods The expression of CD8+CD28- T cells obtained from 50 AS patients and 21 healthy controls were assayed by flow cytometry. CRP was also measured. Results The percentage of peripheral blood CD8+CD28- T cells in patients with AS was significantly increased compared to normal individuals [(18±6)% vs (14±5)%, P=0.020], while the percentage of peripheral blood CD3+,CD8+ CD28+ T cells in patients with AS was significantly decreased[ (65±9)% vs (69±8)%, P=0.039]; [ (15±5)% vs(18±4)%, P=0.038]. No difference was found in CD8+ T cells between patients with AS and normal individuals(P＞0.05). Conclusion The percentage of peripheral blood CD8+CD28- T cells in patients with AS is significantly increased comparing to normal individuals. This suggests that CD8+CD28- T cells may play an important role in the pathogenesis of AS.

Objective To evaluate the clinical effect of ultrasound - guided sclerosing agent lauromacrogol injection in treating lymph leakage. Methods A total of 31 patients with postoperative lymph leakage were selected for this study. Of the 31 patients, successful conservative oppression treatment was accomplished in 16, and lauromacrogol injection had to be carried out in 15 as conservative oppression treatment failed. The patients were followed up and the results were analyzed. Results In 15 patients receiving lauromacrogol injection treatment, complete cure of lymph leak was obtained in 14 with a success rate of 93.33%. Among the 14 cases, the second lauromacrogol injection was employed in 3 at one week after the first injection. Infection occurred in another case one day after the injection , which was cured after dressing change for 15 days. Conclusion For the treatment of lymph leakage, ultrasound-guided sclerosing agent lauromacrogol injection is effective and safe.

Objective To investigate the correlation of short-term systolic blood pressure variability (SBPV) with esti?mated glomeruar filtration rate (eGFR) in the elderly. Methods In physical examination for the third time of kailuan group, the method of cluster sampling was used to collect randomly retired employees, age≥60 in kailuan group. The 24-hour am?bulatory blood pressure monitoring was given to these objects. Finally, 1 405 participants with integral data were recruited in?to the survey. SBPV indices were standard deviation of systolic blood pressure (SD), variability independent of the mean (VIM), maximum-minimum difference (MMD), and average real variability (ARV). Multivariate stepwise linear regression models were used to analyze the influence of short-term SBPV on eGFR. Results (1) Among 1 405 participants (67.16 ± 5.82) years, 933 individuals (66.4%) were male and 472 (33.6%) were female. (2) Study population were divided into four groups based on the 24-hour mean SBP, daytime mean SBP, night time mean SBP (group 1:mean SBP<120 mmHg, group 2:120≤mean SBP<140 mmHg, group 3:140≤mean SBP<160 mmHg, group 4:mean SBP≥160 mmHg), respectively. Values of SD, MMD and ARV, but not VIM were increased with increased mean SBP. (3) The participants were grouped according to the median SBPV with between-group comparison of the eGFR. The average eGFR levels were lower in the high 24-hour SB?PV group (SD, VIM, MMD and ARV), day-time SBPV group (SD, VIM, MMD and ARV) and night-time SBPV group (SD, MMD and ARV) than those in the low SBPV groups (P<0.05). (4) Multivariate stepwise linear regression showed that eGFR increased with 3 indices of 24-hour SBPV (SD, MMD and ARV) and 2 indices of day-time SBPV (MMD and ARV) but not for night-time SBPV (β=-0.07,-0.11,-0.07,-0.12 and-0.07, respectively). Conclusion There is a certain degree of asso?ciation between short-term SBPV indices and eGFR.

Objective To investigate the correlation of the supine hypertension (SP) with carotid intima-media thickness (IMT) in the elderly. Methods Kailuan study is a functional community-based cardiovascular risk factor study. From June 2006, there was a physical examination every two years. In the examination, demographic data, smoking, drinking, physical exercise situation and medication situation were recorded. Levels of triglyceride, high sensitivity C-reactive protein, low density lipoprotein and other biochemical indexes were observed. Using cluster random sampling, 3 064 retired employees of 60 years of age or older were selected. A total of 2 464 subjects took part in an additional examination, including the 24-hour ambulatory blood pressure monitoring, brachial-ankle pulse wave velocity, blood pressure of different positions and urine albumin. Multiple linear regression was used to analyze the correlation between supine systolic blood pressure (SBP) and IMT. Multivariate Logistic regression was used to analyze the effect of SP on IMT. Results (1) Among 2 220 participants (67.29±6.09) years, 1 463 (65.9%) individuals were male and 757 (34.1%) were females, and the average IMT was (0.92 ± 0.18) mm. (2) There was a positive correlation between supine SBP and IMT (r=0.175, P<0.01). (3) After adjusting the confounds, supine SBP was significantly associated with IMT, with an increase of 1 SD (+20.42 mmHg, 1 mmHg=0.133 kPa) in SBP corresponding to an increase of IMT by 0.01 mm (P<0.01). (4) Multiple Logistic regression analysis showed that after adjusting for sitting SBP, age, gender and other factors, SP was still a risk factor of increased IMT (OR=1.37, 95%CI:1.03-1.80), and independent of sitting SBP. Conclusion The supine hypertension is a risk factor of increased IMT, and independent of sitting SBP.

ObjectiveTo detect the levels of bone morphogenetic protein-2(BMP-2) mRNA in gunshot fracture healing of the rabbits.MethodsA model of primary treating gunshot fracture of the rabbit with external fixator and the real time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-RT-PCR) technology with SYBR Green I were established.The levels of BMP-2 mRNA 1,2,3 and 4 weeks after operation were detected with FQ-RT-PCR respectively.ResultsIn the control group,BMP-2 mRNA of the bone tissue began to rise at the first week after fracture and the peak of mRNA expression appeared at the second week.The expression returned to normal at the forth week.In the experimental group,the BMP-2 mRNA rose more slowly that it arrived peak stage at the third week and was significantly lower in experimental group than that in control group at the same phases.ConclusionReal time FQ-RT-PCR is a good method for measuring the levels of BMP-2 mRNA during gunshot fracture healing.The mRNA expression rose more slowly and was significantly lower in gunshot fracture than in common fracture at the same phases.

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.
Animals ; Cell Line ; Genetic Vectors ; genetics ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Porcine respiratory and reproductive syndrome virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, DNA ; genetics ; immunology ; Vaccinia virus ; classification ; genetics ; metabolism ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Matrix Proteins ; biosynthesis ; genetics

UNLABELLEDA novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted.

CONCLUSIONSdouble expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.

Cell Line ; Cloning, Molecular ; DNA, Recombinant ; genetics ; Escherichia coli Proteins ; genetics ; Genetic Vectors ; genetics ; Pentosyltransferases ; genetics ; Porcine respiratory and reproductive syndrome virus ; genetics ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; genetics ; Viral Matrix Proteins ; genetics

A systematic analysis was made on the correlation between acupoints and pathological changes of the zang-fu organs through reviewing of literatures in the database of CNKI from 1959--2011 and the database of Pubmed of the past 10 years. The result showed that specificity was found on the pathological changes of zang-fu organs when acupoints were stimulated. And the pathological changes of the internal organs can be perceived from acupoints on the correspondent meridians, mainly regarding the heart, the stomach, the intestines, the lung and the liver, etc. And most of the researches focused on the correlation between acupoints of the heat meridian and the heart. It was also discovered that a lot of acupoints on various meridians could manifest the pathological changes of the same organ. Different effects of the same acupoints on different times could be found on the same internal organ. Therefore, it is concluded that there is a relative specificity between acupoints and pathological changes of the zang-fu organs. However, it is worth to study the regularity of specificity further.
Acupuncture Points ; Acupuncture Therapy ; Clinical Trials as Topic ; Heart ; physiopathology ; Humans ; Liver ; physiopathology ; Lung ; physiopathology ; Meridians ; Stomach ; physiopathology

AIM:To explore the effect of microRNA-296-5p(miR-296-5p)on neurological damage after cere-bral infarction(CI)and its regulatory relationship with angiotensin-converting enzyme 2(ACE2)signaling pathway medi-ated proliferation of endothelial progenitor cell(EPC).METHODS:Serum samples from 70 patients diagnosed with CI and accompanied by neurological damage in our hospital(CI group)and 70 healthy volunteers(healthy group)were se-lected.The mRNA expression of miR-296-5p,ACE2,and Mas in the serum of both groups were detected by RT-qPCR.The rat model of CI was constructed and SD rats were randomly divided into healthy control group,model control group,sh-miR-296-5p group,and ACE2 overexpression group(OE-ACE2 group).Neurological severity scores(NSS)score was evaluated.The CI status of rats in each group was observed by TTC staining.The mRNA expression of miR-296-5p,ACE2,and Mas in serum of rat was detected by RT-qPCR.EPC were isolated and cultured routinely,and were randomly divided into control group,sh-miR-296-5p group,OE-ACE2 group,OE-miR-296-5p+OE-ACE2 group,and sh-miR-296-5p+sh-ACE2 group.The viability of EPC was detected by CCK-8.Apoptosis of EPC was detected by flow cytometry.The mRNA expression of miR-296-5p,ACE2,and Mas in EPC was detected by RT-qPCR.The relationship between miR-296-5p and ACE2 was verified by dual luciferase reporter gene assay.RESULTS:(1)Clinical trial:compared with the healthy group,the level of miR-296-5p in serum of CI patients was obviously increased(P<0.05),while the mRNA ex-pression levels of ACE2 and Mas were obviously reduced(P<0.05).(2)Animal experiments:compared with the healthy control group,the NSS score,CI area,the level of miR-296-5p in serum,and the mRNA expression level of Mas in the model control group were obviously increased(P<0.05),while the mRNA expression level of ACE2 was obviously de-creased(P<0.05).Compared with the model control group,the NSS score,CI area,the level of miR-296-5p in serum,and the mRNA expression level of Mas in the sh-miR-296-5p group and OE-ACE2 group were obviously reduced(P<0.05),while the mRNA expression level of the ACE2 was obviously increased(P<0.05).(3)Cell experiment:Com-pared with the control group,the A450 and the level of miR-296-5p of EPC cells in the sh-miR-296-5p group and OE-ACE2 group were obviously reduced(P<0.05),the apoptosis rate,the mRNA expression level of ACE2,and Mas were obvious-ly increased(P<0.05).Compared with the sh-miR-296-5p group,the A450 and the level of miR-296-5p in the sh-miR-296-5p+sh-ACE2 group were obviously increased(P<0.05),the apoptosis rate,the mRNA expression level of ACE2,and Mas were obviously reduced(P<0.05).Compared with the OE-ACE2 group,the level of A450 and miR-296-5p in OE-miR-296-5p+OE-ACE2 group were obviously increased(P<0.05),the apoptosis rate,the mRNA expression level of ACE2,and Mas were obviously reduced(P<0.05).CONCLUSION:Knockdown of miR-296-5p may inhibit EPC proliferation by mediating the ACE2 signaling pathway,and alleviate neurological damage after CI.