The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA
Animals ; CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics* ; Gene Editing ; RNA, Guide/genetics* ; Swine

Objective:To analyze the mutations in desmosomal protein genes of arrhythmogenic right ventricular cardiomyopathy (ARVC) in relatives of Yunnan unexplained sudden death (YUSD) cases in Nanjian County, Dali Prefecture, Yunnan Province, and provide a basis for etiological hypothesis and control measures.Methods:The blood samples of YUSD case relatives ( n = 7) and control villagers ( n = 7) were collected, and basic situation investigation and electrocardiography (ECG) examination were performed at the same time. Blood DNA was extracted as a template for PCR amplification, and Sanger method was used to perform plakophilin 2 (PKP2), desmoglein 2 (DSG2), desmocollin 2 (DSC2), desmoplakin (DSP), and junction plakoglobin (JUP) five ARVC desmosomal protein genes sequencing of a total of 97 exons, and comprehensive analysis of gene mutations was carried out. Results:Five of YUSD case relatives carried genetic mutation sites, including DSP gene heterozygous synonymous mutations about exon 20 c.2862 C>T (p.Cys954Cys) and exon 24F c.7122 C>T (p.Thr2374Thr), DSC2 gene heterozygous missense mutation about exon 15 c.2326 A>G (p.Ile776Val), and all the five people were single heterozygous mutation carriers. Among them, two case relatives of the father-son carried the same site mutation of the DSC2 gene; the abnormal ECGs of three YUSD case relatives were ST-T change or clockwise rotation. However, the mutation sites of PKP2, DSG2, DSC2, DSP and JUP genes in control villagers were not detected.Conclusions:YUSD case relatives in Nanjian County carry ARVC desmosomal protein genes DSP and DSC2 mutations. Pathogenic mutation of DSC2 gene c.2326 A>G (p.Ile776Val) is may related to the incidence of some YUSD cases.

Objective To introduce a new minimally invasive lumbar positioning system and its positioning method as well as conduct a prospective control study on the differences in positioning time,positioning frequency and radiation exposure dose between the new positioning method and the traditional positioning method.Methods 121 patients with lumbar disc herniation combined with lumbar instability and patients with thoracolumbar vertebral fracture admitted in our hospital from May 2010 to February 2013 were randomized into two groups.68 patients in Group A had undergone the traditional positioning method before and during operation.Among the 68 patients,41 were with single-segment lesion and 27 were with two-segment lesion.53 patients in Group B had undergone the new minimally invasive lumbar positioning system before and during operation.Among the 53 patients,34 were with single-segment lesion and 19 were with two-segment lesion.There were no significant differences in gender,age,course of disease and other general information between the two groups.The positioning time,positioning frequency and radiation dose of the two groups before and during operation were compared and statistically analyzed.Results The positioning time of Group A and Group B were 8.26±3.44 min and 3.51±1.82 min respectively; the positioning frequencies were 3.57 and 1.22; the accuracy were 60.8％ and 96.2％.For the patients with single-segment lesion,the positioning time before implanting two percutaneous pedicle screws during operation were 15.12±4.69 min and 5.51±1.32 min respectively and the positioning frequency were 6.47 and 2.45.For the patients with two-segment lesion,the positioning time before implanting three percutaneous pedicle screws during operation were 24.91±7.43 min and 8.84±2.32 min respectively and the positioning frequency were 11.72 and 3.69.Moreover,the radiation dose detected at neck,chest and wrist of the surgical staff in Group A were 3.09±0.24 Gy,4.23±0.71 Gy and 5.17±0.62 Gy and that detected in Group B were 1.38±0.47 Gy,2.69±0.33 Gy and 3.21±1.05 Gy.There were significant differences in positioning time,positioning frequency and radiation dose between Group A and Group B.Conclusion The minimally invasive spine positioning system and positioning method can simplify the operative procedures and largely reduce radiation exposure,which is characterized by high positioning accuracy,short positioning time,low fluoroscopy frequency and X-ray radiation dose.

Objective: To observe the clinical efficacy of tuina combined with electroacupuncture (EA) in treating lateral humeral epicondylitis. ＜br> Methods: Totally 118 patients were randomly allocated into a treatment group and a control group, 59 cases in each. The patients in the treatment group were treated with tuina combined with EA treatment, while those in the control group were treated by EA alone. Clinical efficacies of the two groups were compared after the treatment. ＜br> Results: The total effective rate was 93.2% in the treatment group, versus 84.7% in the control group, and the difference was statistically significant (P＜0.05). ＜br> Conclusion: Tuina combined with EA treatment for lateral humeral epicondylitis has a significant effect.

BACKGROUND: Electro-acupuncture therapy shows good central and peripheral analgesic effects. Several studies have shown that transforming growth factor β (TGF-β) plays an important role in the pathogenesis of knee osteoarthritis. OBJECTIVE: To investigate the mechanism underlying electro-acupuncture treatment of knee osteoarthritis. METHODS: A total of 80 healthy male 3-month-old Sprague Dawley rats were randomly and evenly divided into normal, model, electro-acupuncture and drug groups. Rat models of knee osteoarthris were estbalished by ligating the femoral veins and forcing rats to do activies. At 1 month after knee osteoarthris induction, the electro-acupuncture group rats received electro-acupuncture therapy at two acupoints Neixiyan (EX-LE4) and Dubi (ST 35) with a depth of 0.1 cun (pulse 2 Hz, 20 minutes, once a day). The drug group rats were intraarticularly administerd sodium hyaluronate (0.1 mL/administration, once a week). After 2-week treatment, synovial tissue of the knee joint was harvested to determine the exprssion of TGF-β1, TGF-β1 receptor Ⅰand TGF-β1 receptor Ⅱ. RESULTS AND CONCLUSION: TGF-β1 expression in synovial tissue of the knee joint was significantly increased after knee osteoarthris (P < 0.05), but after electro-accupuncture therepy or sodium hyaluronate treatment, TGF-β1 expression was significantly decreased (P < 0.05), moreover, TGF-β1 receptor Ⅰ, Ⅱ expression was signficantly decreased (P < 0.05). These findings suggest that electro-acupuncture for treatment of knee osteoartheis improves the symptoms of osteoarthris by downregulating TGF-β1 expression, and reduction in TGF-β1 receptor Ⅰ, Ⅱ expression promotes the recovery of knee osteoarthris.

ObjectiveTo explore the ideal minimally invasive surgical method for degenerative lumbar spine stenosis.MethodsFrom March 2008 to August 2010,73 cases with lumbar spinal stenosis underwent minimal invasive surgery were retrospectively analyzed.The patients were divided into different groups by clinical features,imaging manifestations,and concurrent diseases.The minimal invasive surgical methods were chosen according to the classification.Operation time,intra-operative bleeding,and complications were recorded.The lumbar function was evaluated by Oswestry disability index (ODI),and the clinical results were assessed by JOA pre- and postoperatively.ResultsMinimal invasive surgery was completed in all cases,which included 25 cases with bilateral decompression and 48 cases with unilateral approach for bilateral decompression.Twenty-three cases used interbody fusion and percutaneous pedicle screw fixation after decompression.Intraoperative blood loss and operation time were related to surgical methods.One case was complicated with dural tear.Cage was inserted into upper vertebral body during interbody fusion in 1case with osteoporosis,removed the cage and implanted bone fusion.Incisions of 3 cases were poor healing after surgery.The average follow up time was 13 months(range,10-35).JOA score and ODI before surgery were 9.2±5.1 and 62.3％±18.5％ respectively; while 6 months after surgery,JOA score and ODI were 23.5±7.2 and 18.4％±6.4％ respectively.JOA score and ODI showed statistically significant improvements after operation(P＜0.01).Twenty-two cases got solid fusion at the final follow-up.ConclusionTreatment of lumbar spinal stenosis by minimal invasive surgery has satisfactory surgical outcomes,but the proper minimal invasive strategy should be chosen according to specific patients,surgeons and hospitals.

Objective: To observe the clinical effect and infra-red thermogram changes for acute lumbar sprain. Methods: All 295 cases with acute lumbar sprain were randomly divided into an electroacupuncture group (147 cases) and medication group (148 cases). The cases in electroacupuncture group were treated by needling Houxi (SI 3), whereas those in the medication group were treated with Meloxicam tablet. The infra-red thermogram was observed before and after treatment. Results: The recovery and effective rates in the electroacupuncture group were 71.4% and 93.9%, whereas the rates in the medication groups were 42.6% and 87.2% (P＜0.01). The temperature differences via the thermogram in the electroacupuncture and medication groups were 2.52℃ and 0.80℃ (P＜0.01), indicating a significant difference.Conclusion: Both electroacupuncture and medication could obtain significant effect for acute lumbar sprain; however, electroacupuncture obtained a better overall effect and a more substantial temperature-raising effect in the thermogram than medication.

To detect the phosphorylation of potassium channel protein (Kv1.2 and Kv1.5) in rat cardiac muscle cells accurately, we applied the combined method of immunoprecipitation and Western blot in this study. Compared with using Western blot alone, the combination of immunoprecipitation and Western blot displayed high sensitivity to detect the activation of potassium channel proteins. Because of its simplicity, quickness and reproducibility, we find that this method was promising for detecting the phosphorylation of Kv1.2 and Kv1.5 proteins or other potassium channel proteins in rat cardiac muscle cells.
Animals ; Blotting, Western ; methods ; Cell Membrane ; metabolism ; Female ; Immunoprecipitation ; methods ; Kv1.2 Potassium Channel ; analysis ; genetics ; metabolism ; Kv1.5 Potassium Channel ; analysis ; genetics ; metabolism ; Male ; Myocytes, Cardiac ; cytology ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley

Objective To detect the expression of RXR? gene protein in gastric cancer, superficial gastritis, normal gastric mucosa, in order to explore the relationship between the development of gastric cancer, combined with clinical and pathological analysis of its clinical significance. Methods Using immunohistochemical detection RXR? protein in gastric cancer, superficial gastritis, gastric mucosa in the normal expression. Results RXR? protein mainly located in the nucleus of cells, there are a small number of cytoplasmic staining, a brown or yellow granular, in gastric cancer, superficial gastritis, normal gastric mucosa, RXR? protein expression rates were 25.0%, 81.1%, 96.1%; group and the normal mucosa and superficial gastritis, gastric cancer group RXR? expression rate has dropped, there were significant differences (P0.05). The lymph node metastasis and tumor pathology and clinical phases closely related (P

ObjectiveTo observe the growth of BT-325 human glioma cells after interfering volume-regulated chloride channel ClC-2 gene.MethodsTwo expression recombinant vectors of ClC-2 gene were designed and constructed. The primary plasmid, pSUPER.puro-shRNA, and the two recombinant plasmids, pSUPER.puro-shRNA-ClC-21 and pSUPER.puro-shRNA-ClC-22, were transfected into BT-325 cells by LipofectamineTM2000 (Groups: control, PP1 and PP2, respectively). The mRNA expression of ClC-2 gene was detected with reverse transcription polymerasse chain reaction (RT-PCR), the cellular survival was determined with MTT assay, and the cell cycle was measured with flow cytometry (FCM). ResultsClC-2 mRNA expression and the growth of the cells in PP1 and PP2 groups were significantly lower than that of control group. The cell cycle progression was blocked in G1 phase (PP1 and PP2 vs control,P<0.01). ConclusionThe growth of BT-325 human glioma cells is prevented by knockdown of ClC-2 gene expression, which may be one of the novel targets to inhibit growth of human malignant glioma cells.