The model of transplanted colonic SW480 cell line carcinoma in gymnomouse body was set up to observe the effect of octapeptide somatostatin (SMS 201-995,SMS) on the transplanted carcinoma and elucidate its mechanism. Results: the volume, weight, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G2M phase in SMS group and SMS+PG (pentagastrin) group were markedly lower than those in PG group and control group, those of PG group were markedly higher than those in control group.The cell amount of G0/G1 phase in SMS group and SMS+PG group was markedly higher than that in PG group and control group, and that of PG group was markedly lower than that in control group.All these suggested that somatostatin could not only inhibit the growth of transplanted human colonic SW480 cell line carcinoma directly but also inhibit the growthpromoting effect of gastrin on the transplanted carcinoma.The mechanism might be that somatostatin inhibit the synthesis of cAMP, DNA and protein in carcinoma cells, then inhibit the cell growing from G0/G1 phase to S and G2M phases.Our study might provide experimental basis for the homonotherapy with analogue of somatostatin in patients with large intestine carcinoma.
In this study, the role of newcastle disease virus (NDV) combined thermic solidified tumor vaccine in inhibiting growth of tumor and immune control was investigated, and rate of inhibiting tumor and cellular immunity were measured. The results showed that rate of inhibiting tumor in experimental group Ⅰ and Ⅱ were 24.8% and 41.1% respectively; average weight of tumor was significantly lower in both experimental groups than in control group, and activity of natural killing (NK) cells in experimental groups was higher than that in control group (Ｐ＜0.01). This suggests that NDV combined thermic solidified tumor vaccine can inhibit growth of tumor and improve activity of NK cells, and their effects are better than that of NDV.
Objective To study the effect of platelet-rich plasma (PRP) on the survival and quality of fat grafts in the nude mice so as to provide a method and the experimental basis for clinical practice. Methods Fat tissue was harvested from the lateral thigh of a 25-year-old healthy woman and the fat was purified by using saline. The venous blood was taken from the same donor. PRP was prepared by centrifugation (200 × g for 10 minutes twice) and activated by 10% calcium chloride (10 : 1). Then 24 female nude mice [weighing (20 ± 3) g, 5-week-old] were allocated randomly to the experimental group and the control group (12 mice per group). Each subcutaneous layer of two sides of the back (experimental group) was infiltrated with 0.8 mL fat tissue-activated PRP mixtures (10 : 2); the control group was infiltrated with 0.8 mL fat tissue-saline mixtures (10 : 2); 0.14 mL activated PRP and 0.14 mL saline were injected into the experimental group and the control group respectively at 5 and 10 days after the first operation. At 15, 30, 90, and 180 days after the first operation, the samples were harvested for gross and histological observations. Results All nude mice survived to the end of the experiment. No inflammation and abscess formation of the graft were observed. Experimental group was better than control group in angiogenesis, liquefaction, and necrosis. The grafted fat weight and volume in the experimental group were significantly larger than those in the control group at 15, 30, and 90 days (P lt; 0.05); but there was no significant difference between the 2 groups at 180 days (P gt; 0.05). Histological observation showed good morphological and well-distributed adipocytes, increasing vacuoles, few necrosis and calcification in the experimental group; but disordered distribution, obvious necrosis, and calcification in the control group. The necrosis area ratio of the experimental group was significantly lower than that of the control group (P lt; 0.05), and the number of micro-vessels was significantly higher in the experimental group than in the control group at 15 and 180 days (P lt; 0.05). Conclusion The method of repeatedly using the PRP within 180 days in assisting fat grafts can obviously improve the survival and quality.
Objective To investigate the mechanism of vascular stromal fraction (SVF) at the early stage after aspirated fat transplantation. Methods Fat was harvested from 5 cases of women undergoing abdominal liposuction operation, and SVF was isolated. Aspirated fat with (group B) or without (group A) SVF was injected subcutaneously into the back of nude mice, and the grafts were harvested at 1, 3, 5, and 7 days. Graft wet weight was measured; and immunohistochemical method (CD31) was performed and the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were qnantified by Western blot assay. Results The wet weight of transplanted adipose tissue showed an increasing tendency in groups A and B with time, and no significant difference was found between groups A and B (P gt; 0.05). At 1 and 3 days after transplantation, no CD31 positive cells was seen in 2 groups; the CD31 positive cells of group B were significantly more than those of group A at 5 and 7 days (P lt; 0.05), and the CD31 positive cells at 7 days were significantly more than those at 5 days in 2 groups (P lt; 0.05). Western blot test showed that VEGF expression reached peak at 3 days , then decreased gradually; the expression of VEGF protein in group B was significantly higher than that in group A at 1, 3, and 5 days (P lt; 0.05). The expression of HGF protein in groups A and B remained at a high level within 5 days, but it tended to decrease at 7 days, which was significantly higher in group B than that in group A (P lt; 0.05). Conclusion SVF can enhance angiogenesis by secretion of growth factors at the early stage after aspirated fat transplantation.
Objective To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2 (CDMP-2) and transforming growth factor β1 (TGF-β1) which were seeded on poly (lactide-co-glycolide) (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Methods Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro. Then induced myoblasts-PLGA scaffold, uninduced myoblasts-PLGA scaffold, CDMP-2 and TGF-β1-PLGA scaffold, and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A, B, C, and D, respectively. At 8 and 12 weeks, the samples were harvested for general observation, HE staining and toluidine blue staining, immunohistochemical staining for collagen type I and collagen type II; the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were determined by RT-PCR, the glycosaminoglycans (GAG) content by Alician blue staining, and the compressive elastic modulus by biomechanics. Results In group A, cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks, and had similar appearance and elasticity to normal cartilage tissue at 12 weeks. In group B, few residual tissue remained at 8 weeks, and was completely degraded at 12 weeks. In groups C and D, the implants disappeared at 8 weeks. HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks; no cartilage lacuna formed in group B at 8 weeks. Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line, with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks; no blue metachromatic extracellular matrix was seen in group B at 8 weeks. Collagen type I and collagen type II expressed positively in group A, did not expressed in group B by immunohistochemical staining. At 8 weeks, the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were detected by RT-PCR in group A at 8 and 12 weeks, but negative results were shown in group B. The compressive elastic modulus and GAG content of group A were (90.79 ± 1.78) MPa and (10.20 ± 1.07) μg/mL respectively at 12 weeks, showing significant differences when compared with normal meniscus (P lt; 0.05). Conclusion Induced myoblasts-PLGA scaffolds can stably express chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of menisci.
Objective Col I A1 antisense oligodeoxyneucleotide (ASODN) has inhibitory effect on collagen synthesis in cultured human hypertrophic scar fibroblasts. To investigate the effects of intralesional injection of Col I A1 ASODN on collagen synthesis in human hypertrophic scar transplanted nude mouse model. Methods The animal model of humanhypertrophic scar transplantation was established in the 60 BALB/c-nunu nude mice (specific pathogen free grade, weighing about 20 g, and aged 6-8 weeks) by transplanting hypertrophic scar without epidermis donated by the patients into the interscapular subcutaneous region on the back, with 1 piece each mouse. Fifty-eight succeed models mice were randomly divided into 3 groups in accordance with the contents of injection. In group A (n=20): 5 μL Col I A1 ASODN (3 mmol/L), 3 μL l iposome, and 92 μL Opti-MEM I; in group B (n=20): 3 μL l iposome and 97 μL Opti-MEM I; in group C (n=18): only 100 μL Opti-MEM I. The injection was every day in the first 2 weeks and once every other day thereafter. The scar specimens were harvested at 2, 4, and 6 weeks after injection, respectively and the hardness of the scar tissue was measured. The collagens type I and III in the scar were observed under polarized l ight microscope after sirius red staining. The ultrastructures of the scar tissues were also observed under transmission electronic microscope (TEM). Additionally, the Col I A1 mRNAs expression was determined by RT-PCR and the concentrations of Col I A1 protein were measured with ELISA method. Results Seventeen mice died after intralesional injection. Totally 40 specimens out of 41 mice were suitable for nucleic acid and protein study, including 14 in group A, 13 in group B, and 14 in group C. The hardness of scars showed no significant difference (P gt; 0.05) among 3 groups at 2 weeks after injection, whereas the hardness of scars in group A was significantly lower than those in groups B and C at 4 and 6 weeks (P lt; 0.05), and there was no significant difference between groups B and C (P gt; 0.05). The collagen staining showed the increase of collagentype III in all groups, especially in group A with a regular arrangement of collagen type I fibers. TEM observation indicated that there was degeneration of fibroblasts and better organization of collagen fibers in group A, and the structures of collagen fibers in all groups became orderly with time. The relative expressions of Col I A1 mRNA and the concentrations of Col I A1 protein at 2 and 4 weeks after injection were significant difference among 3 groups (P lt; 0.05), and they were significantly lower in group A than in groups B and C (P lt; 0.05) at 6 weeks after injection, but no significant difference was found between groups B and C (P gt; 0.05). Conclusion Intralesional injection of Col I A1 ASODN in the nude mice model with human hypertrophic scars can inhibit the expression of Col I A1 mRNA and collagen type I, which enhances the mature and softening of the scar tissue. In this process, l iposome shows some assistant effect.
Objective Tissue engineered bone (TEB) lacks of an effective and feasible method of storage and transportation. To evaluate the activity of osteogenesis and capabil ity of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB. Methods Human bone marrow mesenchymal stem cells (hBMSCs) and decalcified bone matrix (DBM) were harvested from bone marrow and bone tissue of the healthy donators. TEB was fabricated with the 3rd passage hBMSCs and DBM, and they were frozen and dried at extremely low temperatures after 3, 5, 7, 9, 12, and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone (FTEB). TEB and FTEB were observed by gross view and scanning electron microscope (SEM). Western blot was used to detect the changes of relative osteogenic cytokines, including bone morphogenetic protein 2 (BMP-2), transforming growth factor β1 (TGF-β1), and insul in-l ike growth factor 1 (IGF-1) between TEB and FTEB. The ectopic osteogenesis was evaluated by the methods of X-ray, CT score, and HE staining after TEB and FTEB were transplanted into hypodermatic space in athymic mouse. Results SEM showed that the cells had normal shape in TEB, and secretion of extracellular matrix increased with culture time; in FTEB, seeding cells were killed by the freeze-dried process, and considerable extracellular matrix were formed in the pore of DBM scaffold. The osteogenic cytokines (BMP-2, TGF-β1, and IGF-1) in TEB were not decreased after freeze-dried procedure, showing no significant difference between TEB and FTEB (P gt; 0.05) except TGF-β1 15 days after culture (P lt; 0.05). The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation, there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score. On the contrary, there was no ectopic osteogenesis in group DBM 12 weeks after operation. HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation. Conclusion The osteogenic activity of TEB and FTEB is similar, which provides a new strategy to preserve and transport TEB.
Objective To study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
摘要:目的:探讨表达吲哚胺2,3二氧化酶(IDO)的KC对同种异体小鼠移植皮片存活时间的影响及其机制。方法:构建BABL/c →C57BL/6的皮肤移植模型,分别于移植术后第2、7、14天输注KC,于移植术后第7天每组各取2只皮瓣行HE染色和TUNEL以检测淋巴细胞浸润和凋亡情况。KaplanMeier对数秩检验对各组进行生存分析。结果:输入表达IDO和FasL的KC能明显延长BABL/c →C57BL/6皮肤移植模型中皮肤移植物的存活时间,1－甲基色氨酸能阻断此效应。IFNγ组皮瓣浸润淋巴细胞的凋亡率较高(Plt;0.05)。结论:表达IDO和FasL的KC在体内能明显延长同种异体小鼠皮片的存活时间,IDO在KC维持外周免疫耐受中发挥重要作用。Abstract: Objective: To investigate kupffer cells(KC) expressing indoleamine 2,3dioxygenase(IDO) on the survival of grafted skin in mouse and its underlying mechanism. Methods: BABL/c skin was transplanted to C57BL/6. Donor KC were injected i.v. at days 2,7, 14 before transplantation. HE and TUNELAP were used to identify infiltrating cells and apoptotic cells in section of skin allografts from 7 days posttransplantation respectively. The survival rate of recipients among groups were analyzed by Logrank test. Results: Injection of KC expressing IDO and FasL from BABL/c mice into C57BL/6 could prolong a skin graft survival from the donor, but 1methyltryptophan could block the effect in vivo. The apoptosis rate of lymphocyte among skin graft in IFNγ group is more than other group(Plt;0.05). Conclusion: IDO and FasLexpressing KC from the donor of mouse can significantly prolong the skin graft survival. IDO may play an important role in KC to induce immune tolerance.
Objective To provide experimental evidence for the clinical application of ischemia therapy to treating pancreatic cancer. Methods After the model of pancreatic transplanted cancer was established in nude mice with orthotransplantation of human pancreatic cancer cell line into the pancreas, the ischemia of the right lobe of the pancreas was induced with ligation of the gastroduodenal, inferior pancreaticoduodenal and dorsal pancreatic arteries. Effects of regional ischemia on the growth of transplanted cancer and the pathomorphology of the transplanted cancer and pericancerous tissue were investigated. Results The transplanted cancer grew slower and its doubling time was longer in the ischemic group than in the control. On the 3rd, 7th and 14th day after operation, the size of transplanted cancer, the proliferative index and protein content of the cancer cells were significantly lower in the ischemic group than in the control (Ｐ＜0.01). Optical microscopy revealed large areas of coagulation necrosis, necrobiotic cells and the infiltration of inflammatory cells. The atrophy of acini, fibrosis and the infiltration of lymphocyte cells were found in pericancerous tissue. Conclusion Regional ischemia can destroy and inhibit the pancreatic transplanted cancer in nude mice effectively. The ischemia changes of pericancerous tissue may be unfavourable for the growth of the pancreatic transplanted cancer.