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 expression of interleukin-18（IL-18）and signal transducers and activators of transcription 5（STAT5）in retina of 4-24-week-old diabetic rats, and explore the potential molecular mechanisms involved in diabetic retinopathy (DR).MethodsRetinal gene expression profile of healthy and 8-week-old diabetic rats was established with restriction fragment differential displaypolymerase chained reaction (RFDD-PCR), and the differences was analyzed by bioinformatics. IL-18 and STAT5 were filtrated as the candidate genes of DR. The expression of IL-18 and STAT5 in retina of diabetic rats with the age of 4, 8, and 24 weeks was observed by semi-quantitative reverse transcriptase-polymerase chain reaction（RT-PCR）.ResultsThe result of RFDD-PCR showed:expression of IL-18 was higher in healthy retina than that in diabetic one; expression of STAT5 was not found in healthy rats but in diabetic ones. The result of RT-PCR showed：compared with the normal, high expression of IL-18 was found in 4-week diabetic retina, reduced in 8-week one, and decreased to the lowest in 24-week one. The expression of STAT5 was not observed in healthy or 4week diabetic retina, but occurred in 8-week one, and increased in 24-week one. ConclusionThe expression of IL-18 and the activation of STAT5 may relate to the occurrance of DR. The expression of IL-18 doesn′t depend on the activation of STAT5. (Chin J Ocul Fundus Dis, 2005,21:258-260)
Purpose To investigate the expression of the interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-alpha;) in epiretinal membranes(ERM) of eyes with proliferative vitreoretinopathy(PVR). Methods Nineteen epiretinal membranes were obtained form eyes undergoing vitrectomy for retinal detachment complicated with PVR and observed by immunohistochemical methods. Results Expression of IL-6 and TNF-alpha; were observed in 12 and 15 membranes respectively with positive staining mostly in extracellular matrix of epiretinal membranes.Only one membrane showed positive to IL-6 intracellularly,and expression for IL-6 and TNF-alpha; simultaneously in membranes. Conclusion The findings indicate that IL-6、and TNF-alpha;might be involved in the development of PVR. (Chin J Ocul Fundus Dis,1998,14:219-221)
In perioperation period, the dynamic changes of solubla interleulcin-2 receptor (sIL-2R) in serum were determined by ELISA in 60 patients with gastric cancer (GC), and then was compared with those of 30 normal individuals and 40 selective patients who necieved common abdominal surgery. Results: At the day before and ten days after operation, the sIL-2R of patients with GC was higher than that of normal individual. But twenty days after operation, the sIL-2R reduced to as normal level. Conclusion: As a immunodepressive index, the sIL-2R of patients with GC was increased obviously, and after radical gastrectomy, it decreased gradually. So by determining sIL-2R, we can evaluate the immunologic function of patientswith GC.
Objective To investigate the effect of interleukin-10 （IL-10） gene transfer on expression of CD44, selectin-E, lymphocyte function associated antigen-1 （LFA-1）, vascular cell adhesion molecule-1 （VCAM-1） in mice heart transplantation rejection. Methods Model of mice cervical heterotopic heart transplantation was set up, 96 mice were divided into three groups with random number table, control group： heart transplantation between C57 mice; transplant group： heart from BALB/C mice transplant to C57 mice; IL-10 group： IL-10 was transfected on BALB/C mice isolated heart for 1 hour, then transplanted to C57 mice. The messenger ribonucleic acid （mRNA） level expression of CD44 ,selectin-E ,LFA-1 ,VCAM-1 and IL-10 were measured by reverse transcription-polymerase chain reaction （RT-PCR） at the 5th day after transplantation. Results The mRNA level expression of CD44, selectin-E ,LFA-1 ,VCAM-1 in transplant group were significantly increased than those in control group （P〈0.01）. The mRNA level expression of CD44, selectin-E, LFA-1 ,VCAM-1 in IL-10 group were significantly decreased than those in transplant group （P〈0.01）. Conclusion IL-10 gene transfer is able to decrease the expression of CD44, selectin-E,LFA-1 ,VCAM-1 and suppress the heart transplantation rejection in mice.
Objective To determine the contents of matrix metalloproteinase 3 (MMP-3) and interleukin 1 (IL-1) in the tissues of the lumbar disc herniation and to investigate their roles in the pathogenesis. Methods The tissues of the herniated lumbar disc were obtained from 30 patients undergoing surgery for persistent radiculopathy from June 2003 to December 2004 and at the same time these samples were divided into the following three experimentalgroups: the bulge group (n=11), the protrusion group (n=9), and the prolapsus group (n=10)，14 males, 16 females, aged 33.64 years. As the control group, 9 lumbar disc specimens were harvested from 9 patients(4 males, 5 females, aged 21-58 years) suffering from bursting fracture of the lumbar spine. The specimens were analyzed by the ELISA method for the contents of MMP-3 and IL-1. Results The contents of MMP-3(14.25±1.32, 19.89±2.97,20.69±2.18 ng/ml in the bulge group, protrusion group and prolapsus group, separately) and IL-1(8.52±0.22， 11.88±0.52，11.90±0.73 pg/ml in the bulge group, protrusion group and prolapsus group, separately) in the experimental groups were significantly higher than those in the control group. The contents of MMP-3 and IL-1 in the protrusion group were not significantly higher than those in the prolapsus group, but they were significantly higher than those in the bulge group(P＜0.01). The contents of MMP-3 had a significant relationship with the contents of IL-1 in the three experimental groups and the control group(P＜0.01). Conclusion The result demonstrates that the tissues of the lumbar disc herniation can produce both MMP-3 and IL-1, which may have an unknown but important relationship with each other.
63 normal human gallbladders (non-stone group) and 47 inflammed cholesterol stone gallbladders(stone group) were assayed for the amount of macrophages(ΜΦ),the levels of tumor necro-sis factor (TNF) and interleukin 1(1L-1).It was found that in stone group,the amount of ΜΦ was significantly higher than in non-stone group(ΜΦ4101.90±295.72 vs 572.13±30.07AU,Plt;0.01).The levels of TNF and 1L-1 released mainly from the MΦ in stone group were also significantly increased in comparison with those in non-stone group(TNF 18.12±2.03 vs 4.45±0.39ng/mg,Plt;0.001;1L-1 102.42±7.84 vs 66.75±9.50u/mg protein,Plt;0.05).These results suggest that the activited ΜΦ and increases of TNF,1L-1 may be closely related to the inflammatory reaction in gallbladders and the formation of cholesterol gallstones.
Objective To explore the effects of asiaticoside on the activation of nuclear factor kappa B ( NF-κB) and cytokines expression in RAW264. 7 cells induced by lipopolysaccharide ( LPS) . Methods RAW264. 7 cells were allocated to 5 groups, ie. a blank group, a model group stimulated by LPS at dose of 10 μg/mL, and three asiaticoside treatment groups stimulated by LPS and different doses of asiaticoside simultaneously. The effects of asiaticoside ( 10 - 7 , 10 - 6 , 10 - 5 mol /mL) on the proliferation of cells were examined by MTT assay. The activation of NF-κB was detected and analyzed by the laser scanning confocal microscope( LSCM) ,meanwhile the concentrations of TNF-α, IL-1, and IL-10 in supernatants were quantified by ELISA. Results MTT assay showed that asiaticoside ( 10 - 7 , 10 - 6 ,10 - 5 mol /mL) had no effects on the proliferation of RAW264. 7 cells. Asiaticoside significantly decreased the activation of NF-κB, downregulated the secretion of TNF-αand IL-1, and upregulated IL-10 secretion in a dose dependent manner. According to LSCM, the ratio of NF-κB activation was ( 3. 5 ±1. 5) % , ( 75. 7 ±9. 1) % , ( 66. 8 ±7. 1) % , ( 58. 9 ±9. 0) % , and ( 40. 1 ±8. 8) % in the blank, model, and asiaticoside( 10 - 7 , 10 - 6 , 10 - 5 mol /mL) treatment groups respectively. The contents of TNF-α in supernatants were ( 171. 12 ±35. 42, 1775. 45 ±193. 97,1284. 63 ±162. 13,1035. 22 ±187. 97, 598. 90 ±107. 73) pg/mL respectively and IL-1 were ( 5. 66 ±0. 98,26. 93 ±3. 48,22. 41 ±2. 84, 17. 05 ±1. 70, 10. 64 ±1. 29) ng/mL respectively, while IL-10 were ( 25. 23 ±2. 17,71. 75 ±8. 31, 82. 82 ±6. 00, 98. 70 ±8. 84, 119. 97 ±9. 13) pg/mL respectively. Conclusion The antiinflammation mechanism of asiaticoside may be mediated by downregulating inflammatory factors throughNF-κB signal pathway and keeping the balance between proinflammatory and antiinflammatory system.
Objective To explore the therapeutic effect of angiotensin-converting enzyme 2( ACE2) on pulmonary edema after sea-water drowning.Methods Twenty-four Wistar rats were randomly divided into 3 groups, ie. a control group, a seawater drowning group, and an ACE2 treatment group. The rats in the seawater drowning group and the ACE2 treatment group were infused sea-water into their lungs. Then the rats in the ACE2 treatment group were intraperitoneally injected with recombinant rat ACE2. All rats were killed at the time point of 3 hours. Rat arterial blood gas was analyzed and wet /dry weight ratio of lung tissue was measured. The IL-8 content in lung tissue was measured with enzyme linked immunosorbent assay. Pathological changes of lung tissue were observed under light microscope. Results Acute lung injury induced by seawater drowning was successfully reproduced in the rats. The PaO2 in the seawater drowning group was significantly lower than that in the control group and the ACE2 treatment group [ ( 52. 34 ±2. 69) mmHg vs. ( 96. 40 ±3. 47) mm Hg and ( 64. 58 ±3. 42) mm Hg, P lt;0. 05] . The lung W/D ratio and IL-8 level in the seawater drowning group were significantly higher than those in the control group and the ACE2 treatment group ( 8. 30 ±0. 24 vs. 4. 49 ±0. 19 and 5. 65 ±0. 25, P lt; 0. 05; 1112. 2 ±40. 02 vs. 440. 39 ± 4. 06 and 858. 56 ±9. 92, P lt;0. 05) . Lung pathological examination revealed hemorrhage and hyaline membrane formation, alveolar and interstitial edema in the seawater drowning group while those changes significantly relieved in the ACE2 treatment group. Conclusion ACE2 treatment has therapeutic effects on acute lung injury induced by seawater drowning.
Objective To construct recombinant adenovirus vector co-expressing human interleukin (hIL)-10 and green fluorescent protein (GFP) for study of the expression of genes of interest in vascular smooth muscle cells （VSMCs）. Methods hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction (PCR), and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1. Kanamycin resistance screeninged for recombinant plasmids, which were linealized with PmeⅠand transformed into BJ5183-AD-1 containing pAdEasy-1 by electroporation after determining the insert’s sequence correct by NotⅠ and XholⅠdigestion， sequencing and basic local alignment search tool (BLAST). Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells. Amplified plasmids were transfected to AD-293 cells for packaging after being linearized with PacⅠ. PCR was used to determine target gene; The titer of the recombinant adenovirus was measured. VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope. hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay (ELISA). Results Recombinant shuttle plasmids contained interest gene. Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with PacⅠ. PCR indicated that the recombinant adenovirus contained interest gene. The titer of recombinant adenovirus was 3×10１0 efu/ml. Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/106 cells. Conclusion The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs, this lays the foundation for the gene therapy of vascular intimal hyperplasia.