Objective To investigate the effect of myoblast transplantation on duchenne muscular dystrophy (DMD) and to explore the method and feasibil ity of applying gene therapy to DMD. Methods Myoblast of C57/BL10 mice were cultured using multiple-step enzyme digestion method and differential velocity adherent technique. The morphology of the cells was observed with inverted phase contrast microscope. The cells at passage 4 were labeled with 5-BrdU. Twenty-four DMDmodel mice (mdx mice: aged 4-6 weeks, male, 13.8-24.6 g) were randomly divided into two groups (n=12 per group): group A, 1 × 106/mL labeled myoblast were injected via ven caudal is twice at an interval of 2 weeks; group B: 1 mL DMEM/F12 was injected in the same manner serving as a control group. The mice were killed 4 weeks after operation and the motor abil ity of the mice was detected by one-time exhaustive swimming before their death. HE staining and immunohistochemistry staining observation for 5-BrdU, desmin, and dystrophin (Dys) were preformed, and the imaging analysis was conducted. Results The primary myoblast could be sub-cultured 5-7 days after culture, providing stable passage and sufficient cells. The time of onetime exhaustive swimming was (60.72 ± 5.76) minutes in group A and (47.77 ± 5.40) minutes in group B, there was significant significance between two groups (P lt; 0.01). At 4 weeks after injection, HE staining showed that in group A, there were round and transparent-stained myocytes and the percentage of centrally nucleated fibers (CNF) was 67%; while in group B, there were uneven muscle fiber with such pathological changes as hypertrophia, atrophia, degeneration, and necrosis, and the percentage of CNF was above 80%. Immunohistochemistry staining revealed that the expression of 5-BrdU, desmin, and Dys was positive in group A; while in group B, those expressions were l ittle or negative. Image analysis result displayed that integral absorbency (IA) value of desmin was 489.70 ± 451.83 in group A and 71.15 ± 61.14 in group B (P lt; 0.05) and the ratio of positive area to thetotal vision area was 0.314 3 ± 0.197 3 in group A and 0.102 8 ± 0.062 8 in group B (P lt; 0.05); the Dys IA value was 5 424.64 ± 2 658.01 in group A and 902.12 ± 593.51 in group B (P gt; 0.05) and the ratio of positive area to the total vision area was 0.323 7 ± 0.117 7 in group A and 0.035 2 ± 0.032 9 in group B (P lt; 0.05). Conclusion Myoblast transplantation has certain therapeutic effect on DMD of mice.
Objective To investigate the relationship between human leukocyte antigen(HLA)-B51 and Behcet′s disease (BD) with uveities. Methods HLA-A and HLA-B antigen of 27 pateints with BD and 30 healthy persons were detected by microly mphocyte toxicity asssay. HLA-B51 allele (HLA-B5101-B5107) in BD patients with positive HLA-B51 antigen and the controls was detected by polymerase chain reaction-sequence specific primer(PCR-SSP). Results The positive rate of HLA-B51 was 63% and 16.7% in BD patients and the controls, respectively (χ2=12.9, P＜0.001, Pc＜0.05,RR=8.5). Uveities was found in 11 out of 27 BD patients with uveitis. No relativity was found between HLA-B51 and uveitis in BD patients(RR=2.07,χ2=0.759,P＞0.25),and weak association was found between HLA=B5101 and uveitis (RR=2.67, χ2=1.395, P＞0.10). Conclusions HLA-B51 might be a susceptible gene for BD, and there was a weak association between HLA-B51(HLA-B5101) and BD patients with uveitis.(Chin J Ocul Fundus Dis，2004，20：203-205)
The effects of various receptor agonists/antagonists on the accumulation of inositol phosphates(InsPs) in cultured rat retinal pigment epitheium (RPE) cells were analysed. The results showed that serotonin and the 5-HT2 agonists such as alpha;-methyl-serotonin, quipazine, and DOI (1-[2.5-dimethoxy-4-iodopheny1]-2-aminopropane) all stimulated InsPs accumulation in rat RPE cells. The serotonin-induced stimulation of InsPs was effectively blocked by ketanserin, a 5-HT2 antagonist, but also attenuated by the active phorbol ester PMA (4ft-phorbol 12-myristate 13-acetate), a potent protein kinase C activator.The data presented provide clear evidence for the presence of 5-HT2 receptors coupled to phosphoinositide metabolism on cultured rat RPE cells. （Chin J Ocul Fundus Dis,1994,10:80-83）
ObjectiveTo detect expressions of Lgr5 and E-cadherin (E-cad) proteins in gastric cancer tissues and analyze their relationships with the clinicopathologic characteristics and prognosis of patients with gastric cancer.MethodsThe expressions of Lgr5 and E-cad proteins in the 69 patients with gastric cancer and adjacent normal gastric mucosa tissues were measured by the immunohistochemical SABC method, and the relationships between the Lgr5 or E-cad protein expression in the gastric cancer tissues and the clinicopathologic characteristics and the survival of patients with gastric cancer were analyzed.ResultsThe expressions of Lgr5 and E-cad proteins were positive in 60 cases (87.0%) and 30 cases (43.5%) of gastric cancer tissues, respectively, and in 5 cases (16.7%) and 30 cases (100%) of adjacent normal gastric mucosa tissues. There was a significant difference in the positive rate of Lgr5 or E-cad protein expression in the different tissues, respectively (Lgr5 protein: χ2=45.814, P<0.001; E-cad protein: χ2=11.249, P=0.001). The positive rates of Lgr5 and E-cad protein expressions in the gastric cancer were related to the degree of differentiation and the depth of invasion. Meanwhile the positive rate of Lgr5 protein expression in the gastric cancer tissue was also related to the lymph node metastasis and Helicobacter pylori infection, while the positive rate of E-cad protein expression was not related to these (P>0.05). The 5-year total survival time had no significant difference in the patients between with positive and with negative expressions of Lgr5 protein (χ2=1.819, P=0.117), which had a significant difference in the patients between with positive and with negative expressions of E-cad protein (χ2=5.814, P=0.016). The positive expression of Lgr5 was negatively correlated with that of E-cad (rs=?0.355, P=0.003).ConclusionsLgr5 protein may get involved in the mechanism of tumor invasion, lymph nodal metastasis, and low differentiation, while no relationship between the Lgr5 protein and prognosis has been confirmed. E-cad protein may get involved in the mechanism of tumor invasion and affect the prognosis of patients.
Objective To detect the expressions of CK5/6 and Ki-67 in breast cancer， and explore the clinical significance. Methods The expressions of CK5/6 and Ki-67 were detected by immunohistochemistry in 162 cases of breast cancer . The correlation between CK5/6 expression and Ki-67 expression and the relationship of the expressions of two factors to the clinicopathologic factors were analyzed. Results There were 12 cases of the triple negative breast cancer 〔negative estrogen receptor （ER）， negative progesterone receptor （PR）， and negative Her-2〕 in 162 patients with breast cancer. The positive rates of CK5/6 and Ki-67 in the breast cancer tissues were 30.9% （50/162） and 65.4% （106/162），respectively. The positive rates of CK5/6 and Ki-67 in the patients with triple negative breast cancer were significantly higher than those of non-triple negative breast cancer 〔CK5/6：75.0% （9/12） versus 27.3% （41/150）， χ2=11.837， P=0.001；Ki-67：100% （12/12） versus 62.7% （94/150）， χ2=6.847， P=0.009〕. The expressions of CK5/6 and Ki-67 in the breast cancer tissues were not associated with age （P＞0.05）， but they were associated with histology grade （P＜0.05）. The expression of CK5/6 wasn’t associated with tumor size and lymph node status （P＞0.05）， but the expression of Ki-67 was associated with them （P＜0.05）. The expression of CK5/6 in the breast cancer tissue had a positive correlation with the expression of Ki-67 in the breast cancer tissue （rs=0.271， P=0.000）. There were 64 cases when the expression of Ki-67 was （＋＋） and （＋＋＋）， the positive rate of CK5/6 was 43.8% （28/64） and it was significantly higher than that when the expression of Ki-67 was （-） and （＋） 〔22.4% （22/98）， χ2=8.233， P=0.004〕. The positive expressions of CK5/6 and Ki-67 in the breast cancer tissues were negatively correlated with the expressions of ER and PR （CK5/6 and ER：rs=-0.446， P=0.000；CK5/6 and PR：rs=-0.370， P=0.000；Ki-67 and ER：rs=-0.518， P=0.000；Ki-67 and PR：rs=-0.515， P=0.000）. The positive expressions of CK5/6 and Ki-67 in the breast cancer tissue were not correlated with the Her-2 expression （CK5/6 and Her-2：rs=0.105， P=0.183；Ki-67 and Her-2：rs=0.068， P=0.393）. Conclusion The expressions of CK5/6 and Ki67 not only can evaluate the basic rules of breast cancer from origin and development， but also they are beneficial to choose the chemotherapy of different patients.
Objective To investigate the effects of 1, 25-( OH) 2D3 on the expression of matrix metalloprotease-9 ( MMP-9) and nuclear factor κB ( NF-κB) activity in a murine model of chronic asthma. Methods BALB/ c mice were sensitized and challenged with ovalbumin to establish chronic asthmatic model. The animals were randomly divided into a control group, an asthma group and a VD group. Lung sections from the mice were stained by HE and Masson’s trichrome, respectively. Morphometric analysis of the stained sections was performed using computerized image analysis system. Nuclear translocation of NF-κB p65 was examined using Western blot. The level of IκBαwas detected with real-time quantitative PCR ( RTPCR) and Western blot. In addition, the expression of MMP-9 in both activity and mRNA level was detected by gelatin zymograph and RT-PCR, respectively. Results Prominent airway remodeling developed in the asthma group, including the inflammatory cell infiltration, subepithelial collagen deposition and increased airway smooth muscle mass. In contrast, 1, 25-( OH) 2D3 attenuated these established structural changes of the airways. Stimulation with OVA induced a 7. 87-fold increase in the MMP-9 activity compared with that in the control group, and 1, 25-( OH) 2D3 treatment only induced a 3. 46-fold increase in the MMP-9 activity compared with that in the control group ( P lt;0. 05) . The mRNA level of MMP-9 in the VD group ( 3.16 ± 0.09) was decreased compared with the asthma group ( 5.74 ±0.13) ( P lt;0.05) , but itwas still higher than that in the control group ( 0.57 ±0.08) ( P lt;0.05) . 1, 25-( OH) 2D3 reduced the nuclear translocation of NF-κB p65 while up-regulated the IκBα level in lung tissue of chronic asthma. Conclusions 1, 25- ( OH) 2D3 can inhibit the NF-κB activity and down-regulate the expression of MMP-9 in lung tissue of chronic asthma, thus alleviating the established chronic asthma-induced airway remodeling.
Objective To observe the genotype distribution of Haemophilus parainfluenzae from patients with acute exacerbations of chronic obstructive pulmonary disease ( AECOPD) and their effects on A549 cells. Methods 80 hospitalized patients with AECOPD in our hospital were enrolled. Haemophilus parainfluezae were collected by sputum culture and genotyped, then inoculated with cell line A549. IL-6 and IL-8 concentrations in the supernatant were detected and cell morphology was observed at different time points. Results The patients were divided into three groups according to their symptoms. 15 Haemophilus parainfuenzae strains were collected and the positive culture rate between type 1 and type 3 COPD patients were statistically different. The concentrations of IL-6 and IL-8 were both significantly higher than control and increased as time passed. 4 genotypes were got by random amplification of polymorphic DNA ( RAPD) . In RAPD Ⅲ group, the IL-8 concentration was higher at 12h and 24h than others. No morphologic change was found in the cells inoculated with Haemophilus parainfuenzae by microscope after fixing. Conclusions Positive culture rate of Haemophilus parainfuenzae was different in different COPD groups according to symptoms. Haemophilus parainfuenzae can stimulate a cytokine response in A549 cells, maybe one of the pathogens of AECOPD, especially the RAPDⅢ type. Haemophilus parainfuenzae is not an intracellular bacteria.
ObjectiveTo introduce the method and preliminary experience of robot-assisted bilateral internal mammary arteries (BIMA) harvesting for off-pump coronary artery bypass grafting (OPCAB) with 5 grafts via left anterolateral minithoracotomy.MethodsBIMA were harvested using the da Vinci robotic surgical system, and the right internal mammary artery (RIMA) was pulled out of the thoracic cavity through right second intercostal space. Intercepting the distal part of the RIMA for the BIMA composite Lima-Rima Y graft and anastomosing the great saphenous vein with remaining RIMA end to end. The Y graft anastomosed with left anterior descending (LAD) branch and diagonal branches (DIAG), artery-vein graft sequentially anastomosed with blunt round branch, left ventricular posterior branch and posterior descending branch.ResultsThe operation succeeded without hemodynamic instability and intra aortic balloon pump (IABP) implantation or cardiopulmonary bypass. The blood flow of Y graft was 24 mL/min, and the blood flow of artery-vein graft was 30 mL/min. Ventilator assistance time was 35 hours, ICU staying time was 62 hours, and postoperative myocardial enzymes increased temporarily. Postoperative coronary CTA showed that all the grafts were patency, and cardiac ultrasound indicated that the heart function was normal. The patient cured and discharged from hospital 7 days after operation.ConclusionRobot-assisted bilateral internal mammary artery harvesting for OPCAB with 5 grafts via left anterolateral minithoracotomy is feasible, which can achieve complete revascularization.
Abstract: Objective To study the impact of different kinds of mechanical circulation support devices on plasma free hemoglobin(FHb). Methods From Mar. 2004 to Dec. 2005, 20 patients received mechanical circulation support in Fu Wai Hospital, who were divided into 4 groups according to the different type of supporting devices. 9 got extracorporeal membrane oxygenation (ECMO) treatment, 8 received BVS5000 left ventricular support, 2 got MEDOS left ventricular support and 1 received AB5000 left ventricular support. Random control group included 9 cardiotomy patients after CPB supporting and 9 patients with offpump coronary artery bypass grafting during the same period. Parameters such as FHb, Tbil, Dbil, Cr and BUN were monitored throughout the supporting term. The results were compared according to the different types of mechanical circulation support devices. Results The elevation of FHb caused by CPB could be decreased to normal within 1d. However, in BVS5000 group, the elevated FHb level decreased to normal till 2 days later. The others mechanical circulation support devices such as ECMO, MEDOS, AB5000 elevated the FHb throughout the whole supporting period. Compared with those in ECMO group, the patients in BVS5000 group had obviously lower level of FHb since the third day after the beginning of supporting. In patients who got ECMO treatment, there was a trend that the elevation degree of FHb was lower in those with support flow rate less than 2.5 L/min. For most patients got devices support, there was also an elevation of Tbil and BUN level during the supporting period. Conclusion Mechanical circulation support devices, such as ECMO, BVS5000, MEDOS and AB5000, can cause red cell destruction in acceptable level. BVS5000 has much smaller impact on cell destruction than others do in postoperative patients.
ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.