Objective To explore the feasibility of allogeneic marrow stromal stem cells(MSCs) as seed cells to construct tissue engineered bone bydetecting the expressions of interleukin 2(IL-2) and IL-2 receptor in rhesus monkeys after implanting these tissue engineered bones.Methods Engineered bones were constructed with osteoblasts which derived from allogeneic MSCs and bio-derived materials in vitro, and then were implanted to bridge 2.5 cm segmental bone defects of left radius in 15 rhesus monkeys as experimental group, bioderived materials only were implanted to bridge same size defects of right radius as control group. Every 3 monkeys were sacrificed in the 1st, the 2nd, the 3rd, the 6th andthe 12th weeks postoperatively and the expressions of IL-2 and IL-2 receptor in blood and graft samples were detected quantitatively by enzymelinked immuneosorbent assay (ELISA).Results There was no significant difference in the contents of IL-2 and its receptor between 2 groups（P＞0.05）. The contents ofIL-2 and its receptor increased from the 2nd week and maintained high level from the 2nd to the 6th week, but decreased after 6 weeks.ConclusionTissue engineered bones constructed with allogeneic MSCs and bio-derived materials show low immunogenicity. Allogeneic MSCs may be used as seed cells to construct tissue engineered bone.
ObjectiveTo investigate the relationship between the nucleotide binding oligomerization domain like receptor protein 3 (NLRP3) inflammasome and inflammatory reaction of venous ulcer of lower extremity.MethodsTwenty-four patients with active venous ulcer of lower extremity (active ulcer group), 24 patients with non exudative venous ulcer of lower extremity as positive control (non-active ulcer group), and 24 patients with traumatic wound as negative control (traumatic-wound group) were enrolled. The clinical data of the three groups were compared, the tissue samples around the wound were harvested, and the expressions of NLRP3 protein were detected by immunohistochemistry among the three groups. Enzyme linked immunosorbent assay (ELISA) was used to detect the IL-1β and IL-18 protein levels, RT-PCR was used to detect the mRNA expressions of apoptosis associated speck like protein containing CARD (ASC), caspase-1, c-Jun N-terminal kinase (JNK), p38, nuclear factor (NF)-κB p65 and NF-κB inhibitor alpha (NF-κB IkBα), and Western blotting was performed to evaluate the level of NLRP3 inflammasome in wound tissues.ResultsThe inflammatory response in the non-active ulcer group and trauma-wound group were milder than that in the active ulcer group. The levels of IL-1β and IL-18 proteins in the active ulcer group were higher than those in the non-active ulcer group and the traumatic-wound group [IL-1β: (146.621±11.597) ng/L vs. (80.967±14.213) ng/L vs. (84.962±19.484) ng/L, F=136.200, P<0.001; IL-18: (119.814±12.788) ng/L vs. (72.899±17.220) ng/L vs. (48.131±10.407) ng/L, F=167.910, P<0.001]. The results of RT-PCR showed that the mRNA expressions of ASC [(0.030±0.012) ng/L vs. (0.021±0.005) ng/L vs. (0.016±0.004) ng/L, F=18.106, P<0.001], caspase-1 [(0.054±0.012) ng/L vs. (0.013±0.009) ng/L vs. (0.018±0.006) ng/L, F=130.372, P<0.001], NF-κB p65 [(0.093±0.015) ng/L vs. (0.038±0.013) ng/L vs. (0.043±0.014) ng/L, F=110.950, P<0.001], NF-κB IkB-α [(0.085±0.015) ng/L vs. (0.078±0.015) ng/L vs. (0.041±0.016) ng/L, F=53.070, P<0.001], and JNK [(0.075±0.018) ng/L vs. (0.042±0.013) ng/L vs. (0.039±0.014) ng/L, F=41.271, P<0.001] in the wound tissues of the active ulcer group were higher than those in the non-active ulcer group and the traumatic-wound group. And the mRNA expression of p38 in the wound tissues of the active ulcer group was lower than that in the non-active ulcer group [(0.050±0.008) ng/L vs. (0.064±0.014) ng/L, P<0.05]. The result of Western blotting showed that the relative expression level of NLRP3 protein in the wound tissues of the active ulcer group was higher than that in the trauma-wound group and non-active ulcer group (0.767±0.272 vs. 0.605±0.212 vs. 0.556±0.183, F=4.804, P=0.012).ConclusionNLRP3 inflammasome is closely related to the wound in venous ulcer of lower extremity and provides a new target to the therapy of venous ulcer of lower extremity.
Objective To study the interaction and mechanism of prostaglandin I2 (PGI2) receptor/thromboxane A2 (TxA2) receptor (IP/TP) and cyclooxygenase-2 (COX-2) in ischemia reperfusion injury after liver transplantation of rat. Methods Rats were randomly divided into three groups: control group （n=16), orthotropic liver transplantation group （n=32) and nimesulide intervention group （n=32). The samples were obtained at 3 h, 6 h, 12 h and 24 h after operation. The expressions of COX-2, IP and TP mRNA were detected by RT-PCR. Immunohistochemistry was used to detect the localization and expression of COX-2. Hematoxylin Eosin staining was used to classify the injury extent of liver. Serum ALT and AST levels were detected to evaluate the changes of liver enzyme. Results COX-2 protein expression detected by immunohistochemistry in orthotropic liver transplantation group mainly distributed in the district of liver sinusoidal endothelial cells, liver cells and macrophage cells, which was significantly higher than control group and nimesulide intervention group. Expressions of IP mRNA, TP mRNA and COX-2 mRNA in the orthotropic liver transplantation group were significantly increased than those in control group (P＜0.05), and the ratio of IP/TP increased (P＜0.05). Expressions of IP mRNA and TP mRNA in nimesulide intervention group were significantly lower than that in the orthotropic liver transplantation group at 6 h and 12 h after operation (P＜0.05), and the ratio of IP/TP decreased at 3 h, 6 h and 24 h after operation (P＜0.05). The expression of COX-2 mRNA in nimesulide intervention group was significantly lower than that in the orthotropic liver transplantation group at 6 h, 12 h and 24 h after operation. In orthotropic liver transplantation group liver injury was obvious by HE staining, and more severve than that in nimesulide intervention group. Serum AST (each time) and ALT (3 h, 6 h and 12 h) levels in the orthotropic liver transplantation group were significantly higher than that in control group and nimesulide intervention group (P＜0.05) and peaked at 6 h after operation. Conclusion The balance of IP/TP takes part in and plays an important role in the ischemia reperfusion injury of liver transplantation. Changing imbalance of IP/TP may reduce liver transplantation ischemia reperfusion injury by inhibiting COX-2 expression.
Objective In order to study the mechanism of cholesterol gallstone formation through rabbit model which was induced by high cholesterol diet (HCD)Methods the activities of the high density lipoprotein receptor (HDLR) and low density lipoprotein receptor (LDLR) of hepatocytes were investigated. Results The results were as follows: The HDLR activity increased significantly after taking HCD for one week, at the same time, the LDLR activity only increased slightly. Thereafter, the activities of HDLR and LDLR all decreased markedly. As the time of animals taking HCD went on, serum total cholesterol, LDL cholesterol and hepatic cholesterol increased, but bile acids of biliary tract decreased gradually. Conclusion The results suggest that the changes of HDLR and LDLR activities of hepatocytes had no significant effect on bile cholesterol and the decreased HDLR and LDLR activities may cause the reduction some of substrate for bile acids synthesise and play an important role in the formation of gallstone.
Objective To investigate the expression of RNP（alpha defensins） in guinea pigs with bronchial asthma and the influences of P2Y2 receptors on the expression. Methods Seventy-two adult male guinea pigs were randomly divided into a control group （group A） and an asthma group （group B）. The asthma model was established by sensitizing with ovalbumin injection intraperitoneally and challenging with ovalbumin inhalation. Then the A and B groups were divided into following subgroups,ie,the saline groups （A1/B1），ATP groups （A2/B2），and Suramin groups （A3/B3），in which the animals were intervented with saline，ATP，and suramin respectively. Total pulmonary resistance （RL） was measured.Total and differential cell counts in bronchoalveolar lavage fluid （BALF） were measured. Pathological changes of lung were observed under light microscope using HE staining. The plasma levels of RNP and IL-8 were measured by ELISA. The protein and mRNA expressions of RNP and P2Y2 were detected by immunohistochemistry and RT-PCR. Results The RL increased significantly as much as 10% in group B compared with group A. Pathological study revealed that luminal narrowing of bronchus and inflammatory cells infiltration in the asthma group. The total cell and polymorphonuclear neutrophil （PMN） counts in BALF in group B1 were significantly higher than group A1 and B3，but lower than group B2 （Plt;0.05）,but no significant difference among group A1，A2，or A3 was found. The RNP expression in plasma and lung tissue in group B1 was higher than group A1 and B3，but lower than group B2 （Plt;0.05）. The RNP expression in plasma and lung tissue was lower in group A1 than group A2，but higher than group A3（Plt;0.05）. The IL-8 expression in plasma in group B1 was significantly higher than group A and B3，but lower than group B2 （Plt;0.05） but no significant difference among group A1，A2，or A3 was found（Pgt;0.05）. RNP protein expressed predominantly in lung tissues，especially where the PMN infiltrated. The expression of RNP mRNA were consistent with the RNP protein expression in all groups. P2Y2 mRNA expression in group A1 was lower than group A2，and higher than group A3 （Plt;0.05）.P2Y2 mRNA expression in group B1 was lower than group B2 but higher than group B3 （Plt;0.05）. The linear correlation analysis showed that RNP had no significant correlation with PMN and IL-8 in group A，and was positively correlated with P2Y2 mRNA （Plt;0.05） while RNP was positively correlated with PMN，IL-8，and P2Y2 mRNA in group B. Conclusion RNP expression increases in bronchial asthma guinea pigs and may be related to P2Y2 receptors.
【Abstract】 Objective To investigate the expression and significance of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic cancer. Methods Thirty-two samples of pancreatic cancer tissue were collected from year 2002 to 2004. All of them were verified by histopathology and there were 9 cases of well-differentiated, 12 of moderately differentiated, and 11 of poorly differentiated, in which 12 cases were in the stage of Ⅰor Ⅱand 20 in the stage of Ⅲ or Ⅳ according to the TNM staging method. Eighteen normal pancreatic tissues were used as control group. The expressions of TRAIL receptors (death receptor 4, death receptor 5, decoy receptor 4 and decoy receptor 5) mRNA were assayed by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) in the pancreatic cancer tissues and the normal pancreatic tissues. Results The expressions of death receptor 4 (DR4) and death receptor 5 (DR5) were detected in all the pancreatic cancer tissues and the normal pancreatic tissues and the levels of DR4 and DR5 were significantly higher than those of the normal pancreatic tissues (P＜0.01). Decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) were also expressed in normal pancreatic tissues, whereas DcR1 and DcR2 were only expressed in 18 and 20 pancreatic cancer tissues, respectively. However, there were no significant difference of the expression of DcR1 and DcR2 between the pancreatic cancer tissues and the normal pancreatic tissues （Pgt;0.05）. The expression level of DR5 in pancreatic cancer tissue was correlated with tumor differentiation and clinical stage, and the levels in stage Ⅲ and stage Ⅳwere significantly lower than those of stageⅠand stageⅡ(P＜0.05). The expressions of DR4, DcR1 and DcR2 were not correlated with tumor differentiation and clinical stage (Pgt;0.05). Conclusion ①The expression of TRAIL receptors in pancreatic cancer tissues is prevalent, but the types of receptors expressed in different tissues were also different. High expression of death receptors may play an important role in TRAIL recptors regulated pancreatic cancer apoptosis. ②The expression of DR5 is correlated with the differentiation degree of pancreatic cancer cell and clinical stage of tumor. The expressions of DR4, DcR1 and DcR2 should not be considered as related indexes of differentiation degree or clinical stage of pancreatic cancer.
Objective To investigate the inhibitory effects of fms-like typrosine kinase receptor sFlt-1 on retinal neovascularization (RNV).Methods Recombinant lentivirus sFlt-1（2-3）and sFlt-1（2-4）expressing the sFlt-1 (2-3) and (2-4) immunoglobulinlike regions of sFlt-1 were constructed. 96 seven-day-old C57/6J mice were randomly divided into 4 groups with 24 mice in each group. Group 1: normal control; group 2: experimental control; group 3: sFlt-1（2-3）; group 4: sFlt-1（2-4）.The mice in group 2-4 were exposed to hyperoxia with （75plusmn;2）% O2 for 5 days and then returned to normoxia with 21% O2;the mice received an intravitreal injection with 1 mu;l virus of empty vector, sFlt-1（2-3）,or sFlt-1（2-4）,respectively. Five days later, all mice underwent perfusion fluorecein angiography and retinal wholemont was made to observe the changes of retinal vessels; retinal sections were stained by hematoxylin and eosin and RNV endothelium cell nucleus were counted; vascular endothelial growth factor(VEGF) and VEGF receptor-2 (KDR/Flk-1) protein were measured by Western blot.Results Seventeen days after birth, the retinal area of fluorescein leakage and RNV, RNV nucleus which breaking through inner limiting membrane in group 3 and 4 were smaller or less than that in group 2（P＜0.01）; while VEGF protein didnprime;t changed much （P＞0.05）the expression of KDR/Flk-1 decreased significantly (P＜0.01). Conclusion sFlt-1（2-3）and sFlt-1（2-4）can inhibit the formation of oxygen-induced RNV,the former virus has a better effect.
The aim of this study was to establish stable expression of human thyroid stimulating hormone receptor (TSHR) α-subunit (hTSHRA) on human embryonic kidney 293T (HEK 293T). HEK 293T cell lines with stable expression of hTSHRA could be used for detecting affinity between hTSHRA and potential TSHR blocking-peptide. We firstly constructed hTSHRA gene into lentiviral vectors GV218. The sequence comparison indicated that we had constructed GV218-hTSHRAA. Western blot demonstrated the 52 kD aim band of hTHSRA on over-expressed HEK 293T cells. GV218-hTSHRA constructions and pHelper were then co-transfected into HEK 293T cells to form packaging plasmid. The HEK 293T cells that stably expressed hTSHRA could also express green fluorescent protein. The titer of lentiviral packaging vector is 2×108 TU/mL with qPCR. The lentiviral packaging vector thereafter was transfected into HEK 293T cells again. The hTSHRA expressed on the HEK 293T cells. Human TSHRA stably expressed on HEK 293T upon continuously passaging. Therefore, we established hTSHRA stable expression on HEK 293T cells by constructing GV218-hTHSR lentiviral packaging vector. It is a useful tool for studying TSHR affinity with anti-thyroid peptide.
Objective To explore the effects of prolonged inhalation of Aspergillus fumigatus ( Af)spores on epithelial cell injury and expression of epidermal growth factor receptor( EGFR) in airways of asthmatic rats. Methods 64 male Wistar rats were randomly divided into 8 groups, ie. chronic asthma group ( group A) , chronic asthma plus Af spores inhalation for 1 week ( group B) , 3 weeks ( group C) and 5 weeks ( group D) , chronic asthma plus saline inhalation for 5 weeks ( group E) , OVA-sensitized and salinechallenged group ( group F) , and OVA-sensitized and saline-challenged plus Af spores inhalation for 5 weeks ( group G) ( each n =8) . The airway resistance ( Raw) and change of Raw after acetylcholine provocation were detected using a computerized system. The concentrations of epidermal growth factor ( EGF) andtransforming growth factor alpha( TGF-α) in BALF were measured by ELISA. The extents of epithelial cell injury and goblet cell hyperplasia were evaluated on hematoxylin and eosin-stained( HE) and periodic acidschiff ( PAS) stained lung sections. The expression of EGFR in airway epithelia was demonstrated byimmunohistochemistry, and the level of EGFR protein in the rat lung tissues was measured by western blot.Results The concentration of EGF( pg/mL) ( 51. 72 ±8. 54, 68. 12 ±7. 85, 86. 24 ±9. 12, respectively)and TGF-α( pg/mL) ( 55. 26 ±9. 30, 75. 58 ±11. 56, 96. 75 ±14. 66, respectively) , detached/ inner perimeter of epithelium( % ) ( 11. 25 ±3. 12, 26. 45 ±5. 56, 28. 50 ±7. 50, respectively) , the ratio of goblet cell area to epithelial cell area ( % ) ( 16. 42 ±5. 24, 22. 64 ±6. 82, 36. 38 ±9. 21, respectively) , the integrated optical density ( IOD) of EGFR positive stain in airway epithelial cells ( 82 ±15,120 ±19, 165 ±21, respectively) , and the EGFR protein levels in lung tissues ( 0. 91 ±0. 26, 1. 61 ±0. 52, 2. 52 ±0. 78,respectively) in group B, C, and D were higher than those in group A, E, F and G( P lt; 0. 05 or P lt;0. 01) .The change rates of Raw( % ) ( 61. 91 ±5. 26, 84. 69 ±6. 38) in group C and D were higher than those in group A, E, F and G ( P lt; 0. 05 or P lt;0. 01) . The IOD of EGFR was positively correlated with detached/inner perimeter of epithelium( % ) and the ratio of goblet cell area to epithelial cell area( % ) ( r = 0. 692,P lt;0. 01; r = 0. 657, P lt; 0. 01, respectively) . Conclusion Prolonged inhalation of Aspergillus fumigatus spores can aggravate airway epithelial cell injury, up-regulate the expression of EGFR in airway epithelial cell and induce goblet cell hyperplasia, thus increase the airway responsiveness in rats with chronic asthma.