BACKGROUND: Growth differentiation factor 5 (GDF-5) is an important factor to regulate the formation and development of the cartilage and bone, it plays a crucial role on the promotion of repairing bone, cartilage and tendon ligament injury. OBJECTIVE: To transfect eukaryotic expression plasmid pcDNA 3.1(+)/GDF-5 to bone marrow stroma stem cells of mouse and to check the expression of extracellular matrix and proteoglycan which relates with the cartilage formation and differentiation. DESIGN, TIME AND SETTING: An in vitro observation regarding cells was performed in the central laboratory of Wuhan Union Hospital between March and December in 2008.MATERIALS: Twenty Kunming specimen male mice were offered by Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology. The eukaryotic expression plasmid pcDNA 3.1(+)/GDF-5 was preserved at the laboratory.METHODS: The man-ow stroma stem cells were isolated from mouse bone marrow and cultured in vitro with whole bone marrow adherence method. Passage 3 cells were incubated on 6-well plate and began to transfect when they were 90% confluent. Experiment was assigned into three groups: trensfection group underwent transient transfection of liposome-mediated pcDNA 3.1(+)/GDF-5 using LipofectamineTM2000; blank plasmid group was transfected with blank plasmid pcDNA 3.1(+); control group was added with equal volume of liposome and other protocols were the same as above. MAIN OUTCOME MEASURES: The transfection efficacy was identified success by the expression of GDF-5 gene and protein using RT-PCR and immunocytochemistry at 72 heurs following transfection, cartilage matrix Ⅱ collagen expression was determined as above methods. Then marrow stroma stem cells were cultured for additional two weeks to check expression of proteoglycan with alcian blue staining.RESULTS: In the transfection group, a 219-bp specific amplification band was visible, there were brown positive stain in the cytoplasm of marrow stroma stem calls; In blank plasmid group and control group, no GDF-5 trensfection, specific amplification band or obvious stain of cytoplasm was observed. In the transfection group, the collagen Ⅱ gene was detected to express at 225 bp, with brown yellow stain in cytoplasm; in the blank plasmid group and control group, no collagen Ⅱ gene expression or SP ～ stain was observed. Alcian blue staining results showed the transfected cells were stained blue while those in the blank plasmid group and control group were not metachromasia stained.CONCLUSION: Gene trensfection of pcDNA 3.1 (+)/GDF-5 to marrow stroma stem cells can significantly raise expression of collagen II and proteoglycan, and promote the chondrogenic differentiation of marrow stroma stem cells.

BACKGROUND:Overweight or obesity is the most significant risk factors for knee osteoarthritis.Time-restricted diet shows an effective effect in preventing overweight or obesity.Whether infrapatellar fat pad,an important component of the knee joint,is affected by time-restricted diet and thus influences knee osteoarthritis remains unclear. OBJECTIVE:To employ a high-fat diet-induced rat model to investigate the effect of weight loss by time-restricted diet on infrapatellar fat pad,thereby providing evidence for early prevention and treatment of obesity-related knee osteoarthritis. METHODS:Fifteen male Sprague-Dawley rats were randomly divided into three groups(n=5 per group).Rats in the control group were allowed to eat at will for 24 hours and were fed the defined control diet(12%fat);rats in the high-fat diet group were allowed to eat at will for 24 hours and were fed the high-fat diet(45%fat);and rats in the high-fat diet with time-restricted diet group were fed the high-fat diet only from 9:00(2 hours after the light)to 17:00(2 hours before the dark).After 8 weeks of feeding,mDixon-Quant sequence was used to assess proton density fat fraction in the infrapatellar fat pad and subcutaneous adipose tissue in the right inguinal region.ELISA was used to quantify differences adipokine.Sirius red staining was used to evaluate changes in fibrosis of the infrapatellar fat pad.The expressions of uncoupling protein-1 in the infrapatellar fat pad and subcutaneous adipose tissue in the right inguinal region as well as leptin,adiponectin and tumor necrosis factor-α in the infrapatellar fat pad were detected by immunohistochemistry staining. RESULTS AND CONCLUSION:After 8 weeks of feeding,compared with the high-fat diet group,the body mass of rats in the control group(P=0.036)and the high-fat diet with time-restricted diet group(P=0.003)was significantly reduced.The proton density fat fraction in the infrapatellar fat pad in the high-fat diet group was significantly higher than that in the control group(P<0.001)and the high-fat diet with time-restricted diet group(P=0.004),while there was no significant difference in the proton density fat fraction of the subcutaneous adipose tissue among the three groups.The serum leptin levels of rats in the high-fat diet group were significantly higher than those in the control group(P=0.030)and the high-fat diet with time-restricted diet group(P=0.018).Compared with the high-fat diet group,the infrapatellar fat pad fibrosis characterized by Sirius red staining in the control group(P<0.001)and the time-restricted diet group(P=0.003)was significantly decreased.The expression of leptin in IFP of the high-fat diet group was significantly higher than that of the control group(P<0.001)and the high-fat diet with time-restricted diet group(P<0.001).The expression of adiponectin in the infrapatellar fat pad of the high-fat diet group was significantly lower than that of the control group(P=0.004)and the high-fat diet with time-restricted feeding group(P=0.048).However,there was no positive expression of uncoupling protein-1 in the infrapatellar fat pad and subcutaneous adipose tissue of all the three groups,and no positive expression of tumor necrosis factor-α in the infrapatellar fat pad.To conclude,time-restricted diet could retard the fibrosis of the infrapatellar fat pad,reduce the proton density fat fraction of the infrapatellar fat pad,and affect the level of adipokine in serum and infrapatellar fat pad.Time-restricted diet may become a simple and effective option for the treatment and prevention of obesity-related knee osteoarthritis.

Objective To explore the availability and safety of ileal ureter replacement combined with ileal augmentation cystoplasty in treating ureteral stenosis with contracted bladder.Methods From August 2015 to November 2018,three patients who underwent ileal ureter replacement combined with augmentation cystoplasty were treated with ileal ureter replacement combined with augmentation cystoplasty.There were 1 male and 2 females with the age ranging from 34 to 55 years (mean 39 years).Two patients suffered from left ureter stenosis,and one patient had stenosis on the both sides.The length of the ureter stenosis ranged from 6 to 18 cm (mean 9.8 cm).The preoperative bladder capacity ranged from 60 to 150 ml (mean 103.3 ml).In the surgery,part of ileum was used to replace the ureter,and the distal intestine was made into U-shape to enlarge the bladder.Results All operation were completed successfully.The operation time ranged from 220 to 400 min (mean 303.0 min),and the blood loss ranged from 150 to 500 ml (mean 283.3 ml).Laparoscopic surgery was performed in 1 case and open surgery in 2 cases.Three weeks after the surgery,the bladder volume underwent cystography ranged from 300 to 400 ml (mean 360.0 ml).Three months after the surgery,the postvoid residual urine volume ranged from 20 to 50 ml (mean 33.3 ml).Postoperative frequency and urgency of urine were completely relieved in 1 case,alleviated in 2 cases.Flank pain was completely relieved in 1 case,and alleviated in 2 cases postoperatively.The serum creatinine of 2 patients mildly increased after the surgery,while 1 patient remained stable.For complications,1 patient had urinary infection,and 1 patient suffered from metabolic acidosis.Conclusions Ileal ureteral replacement combined with augmentation cystoplasty can be the choice of treatment for long segment ureteral stenosis and enlarge the bladder simultaneously.The postoperative complications and the kidney functions should be regularly followed up.

Objective:To investigate the effect of time-restricted feeding (TRF) on bone marrow fat of proximal femur in obese rats induced by high-fat diet (HFD) using proton density fat fraction (PDFF).Methods:Totally 30 male Sprague-Dawley rats were stratified and randomly sampled into 6 subgroups according to body weight, with 5 rats each. Then 2 subgroups were combined into one group, and there were totally 3 groups. The rats in the control group were fed with normal diet, and the rats could eat as much as they wanted for 24 h; the rats in the HFD group were fed with high-fat diet, and the rats could eat as much as they wanted for 24 h; the rats in the HFD+TRF group were fed with high-fat diet only between 9 AM (2 h after light) and 17 PM. One subgroup of rats from each group was examined with MRI on the femur on day 28 of the experiment, and the other subgroup from each group was examined on day 56 to measure the bone marrow PDFF of proximal femur based on mDixon-Quant quantitative sequence images. The rats were executed at the end of the scanning period, and blood samples were collected to measure serum levels of leptin. One-way ANOVA or Kruskal-Wallis H test was used to compare the differences in body weight, PDFF, and serum levels of leptin among 3 groups. The LSD- t test was used for multiple comparisons. Results:On day 28 of the experiment, the differences in body weight, PDFF, and serum leptin among the 3 groups of rats were not statistically significant ( P>0.05). On day 56, the bone marrow PDFF of proximal femur of the rats in the control group, HFD group, and HFD+TRF group were (7.2±1.4)%, (9.7±2.4)%, and (11.2±3.6)%, respectively. The differences in body weight, PDFF, and serum levels of leptin among the 3 groups of rats were statistically significant ( F=6.95, P=0.010, F=5.98, P=0.007, F=4.54, P=0.034). The results of multiple comparisons showed that the body weight in the HFD group was higher than those in the control group (LSD- t=52.96, P=0.036) and the HFD+TRF group (LSD- t=82.74, P=0.003). The values of bone marrow PDFF of proximal femur in the HFD+TRF group was higher than that in the control group (LSD- t=4.01, P=0.012). The serum levels of leptin in the HFD group were higher than those in the control group (LSD- t=1.45, P=0.030) and the HFD+TRF group (LSD- t=1.62, P=0.018). Conclusion:TRF induces an increase in the values of bone marrow PDFF of proximal femur in conjunction with weight loss in obese rats induced by HFD, and the increase in bone marrow fat may be related to the decrease in serum leptin.

ObjectiveTo explore the effectiveness and safety of Shenlong Dingji Formula （参龙定悸方） on the quality of life in patients with paroxysmal atrial fibrillation （PAF） of qi-yin deficiency and phlegm-stasis obstructing collaterals syndrome. MethodsA total of 60 patients with PAF of qi-yin deficiency and phlegm-stasis obstructing collaterals syndrome were recruited and randomly divided into a treatment group and a control group， with 30 patients in each group. The control group received standard western medicine treatment， while the treatment group was additionally given Shenlong Dingji Formula orally， one dose per day. Both groups were treated for 4 weeks. The primary outcome measure is the Atrial Fibrillation Effect on Quality of Life （AFEQT） score including scores of four dimensions，i.e. atrial fibrillation-related symptoms， treatment concerns， daily activities， and treatment satisfaction. The secondary outcome measures included the frequency and duration of symptomatic atrial fibrillation episodes and traditional Chinese medicine （TCM） syndrome scores covering symptoms such as palpitations， chest tightness， fatigue， shortness of breath， reluctance to speak， spontaneous sweating， stabbing pain， and insomnia. These indicators were assessed at baseline （before treatment）， after 2-week of treatment， after 4-week of treatment， and 4 weeks after the end of treatment （follow-up）. Additionally， safety indicators before and after treatment and adverse events occurring during the trial were recorded to evaluate safety. ResultsA total of 56 patients completed the study， with 28 in each group. Primary outcome indicators： 1） the treatment group showed significant improvement in the total score of the AFEQT scale， with significantly higher total scores after 2-week treatment， 4-week treatment， and follow-up compared to the previous time point （P＜0.05）. In the control group， the AFEQT score significantly increased only after 4-week treatment compared to baseline （P＜0.05）. In the treatment group， the AFEQT scores after 2-week， 4-week treatment， and during follow-up were all higher than those of the control group at the corresponding time points （P＜0.01）. 2） In the treatment group， there was no statistically significant difference in the AFEQT treatment satisfaction dimension score during follow-up compared to that after 4-week treatment （P＞0.05）. However， the scores for all other dimensions at each time point were higher than those at the previous time point （P＜0.05）. In the control group， the scores for the atrial fibrillation-related symptom dimension were higher after 2-week and 4-week treatment than those of the previous time points （P＜0.05）. For the treatment satisfaction dimension， significant increases were observed only after 2-week and 4-week treatment compared to baseline （P＜0.05）. Secondary outcome indicators： 1） In the treatment group， the frequency and duration of symptomatic atrial fibrillation episodes decreased significantly at each time point compared to the previous time point （P＜0.05）， except for the duration of trial fibrillation at follow-up. In the control group， the frequency of episodes decreased significantly at all time points compared to baseline （P＜0.05）， while the duration of trial fibrillation showed a significant reduction at follow-up compared to those after 2-week treatment （P＜0.05）. 2） In the treatment group， TCM syndrome scores significantly reduced after 2-week treatment， 4-week treatment， and during follow-up compared to the previous time point and baseline （P＜0.05）. In the control group， significant reductions were observed only after 4-week after treatment and during follow-up （P＜0.05）. The TCM syndrome scores in the treatment group were lower than those in the control group at the same time points （P＜0.01）. No adverse events occurred during the trial in either group， and safety indicators showed no significant changes after treatment. ConclusionShenlong Dingji Formula effectively improves the quality of life， alleviates TCM syndromes， and reduces the frequency and duration of symptomatic atrial fibrillation in patients with PAF of qi-yin deficiency and phlegm-stasis obstructing collaterals syndrome， and demonstrates good safety.

Purpose To explore the value of contrast-enhanced ultrasound(CEUS)in the differential diagnosis of gallbladder polypoid lesions(GPLs)(diameter≥10 mm).Materials and Methods A prospective enrollment of 229 patients with GPLs who underwent cholecystectomy in 17 hospitals from December 1 2021 to June 30 2024 was conducted to analyze the relationship between general data,conventional ultrasound,CEUS characteristics and the nature of GPLs.Multivariate Logistic regression was employed to identify independent risk factors for neoplastic polyps,the differential diagnostic value of different indicators was compared.Results Among 229 patients with GPLs,there were 108 cases of cholesterol polyps,102 cases of adenoma and 19 cases of gallbladder cancer.Age(Z=-4.476,P<0.001),polyp number(χ2=15.561,P<0.001),diameter(Z=-8.149,P<0.001),echogenicity(χ2=9.241,P=0.010),vascularity(χ2=23.107,P<0.001),enhancement intensity(χ2=47.610,P<0.001),enhancement pattern(χ2=6.468,P=0.011),vascular type(χ2=84.470,P<0.001),integrity of gallbladder wall(χ2=7.662,P=0.006)and stalk width(Z=-9.831,P<0.001)between cholesterol polyps and neoplastic polyps were statistically significant.Age,location,diameter,echogenicity,enhancement pattern,vascular type and stalk width between adenoma and gallbladder cancer were statistically significant(Z=-4.333,-3.902,-5.042,all P<0.05).Multivariate Logistic regression analysis showed that hyper-enhancement,branched vascular type and stalk width were independent risk factors for neoplastic polyps(OR=4.563,5.770,3.075,all P<0.001).The combination of independent risk factors was better than single factor and diameter in the differential diagnosis of cholesterol polyps and neoplastic polyps(all P<0.01).Conclusion CEUS can effectively identify the nature of GPLs and provide a valuable imaging reference for the selection of treatment methods.