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.
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.
【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
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 provide the seed cells for bone tissue engineering, to establ ish immortal ized human bone marrow mesenchymal stem cells (MSCxj) and to investigate the ectopic osteogenesis of MSCxj. Methods MSCxjs of the 35thand 128th generations were maintained and harvested when the cell density reached 2 109. Then, these cells were co-cultured with heterogeneous bone scaffold in groups A (the 35th generation, n=12) and group B (the 128th generation, n=12); heterogeneous bone alone was used in group C (n=12). The cell prol iferation was observed by scanning electron microscopy (SEM) after 48 hours and 18 days of osteogenic induction culture. The complex was implanted subcutaneouly through a 3-mm-incision at both sides of the back in 18 nude mice. Tetracycl ine label ing was performed before the animals were sacrificed. Tetracycl ine fluorescence staining, HE staining, ponceau staining, and immunohistochemistry staining for osteocalcin were performed at 4, 8, and 12 weeks after transplantation; the morphologic quantitative analysis was made. Results After 48 hours, SEM showed that MSCxjs adhered to heterogeneous bone and grew well; after 18 days, a large number of new filamentous extracellular matrix and small granules were found to cover the cells. The results of tetracycl ine fluorescence staining, HE staining, and ponceau staining in groups A and B showed that the osteogenesis was not obvious at 4 weeks after transplantation; osteoid matrix deposition was noted around and in theheterogeneous bone at 8 weeks; and osteogenesis was increased at 12 weeks. There was no significant difference in bone formation between groups A and B. Osteogenesis was not observed in group C. The osteocalcin expressions were positive in groups A and B. The bone ingrow percentages of groups A and B were 5.64% ± 2.68% and 4.92% ± 2.95% at 8 weeks, and 13.94% ± 2.21% and 14.34% ± 3.46% at 12 weeks, showing significant differences between 8 weeks and 12 weeks at the same group (P lt; 0.05) and no significant difference between groups A and B at the same time (P gt; 0.05). Conclusion MSCxj has favorable abil ities of ectopic osteogenesis and can be appl ied as seeded cells in bone tissue engineering.
Objective To investigate the feasibility and characteristic of tissue engineered testicular prosthesis with highdensity polyethylene(HDPE，trade name: Medpor) and polyglycolic acid(PGA). Methods The chondrocytes were isolated from the swine articular.The PGA scaffold was incorporated with medpor which semidiameters were 6mmand 4mm respectively.Then, the chondrocytes (5×10 7/ml) were seeded onto Medpor-PGA scaffold and cultured for 2 weeks. The ten BALB／C mice were divided into two groups randomly(n=5). In the experimental group, the cell-scaffold construct was implanted into subcutaneous pockets on the back of nude mice. In the control group, the Medpor-PGA scaffold was implanted. The mice of two groups were sacrificed to harvest the newly formed cartilage prosthesis after 8 weeks. Macroscopy, histology and immunohistochemistry observations were made. Results The gross observation showed that on changes were in shape and at size, the color and elasticity were similar to that of normal cartilage and that the cartilage integrated with Medpor in the experimental group; no cartilage formed and fiberlike tissue was found in the control group. HE staining showed that many mature cartilage lacuna formed without blood vessel and some PGA did not degradated completely. Toluidine blue staining showed extracellular matrix had metachromia. Safranin O-fast green staining showed that many proteoglycan deposited and collagen type Ⅱ expression was bly positive. In the control group, Medpor was encapsulated by fiber tissue with rich blood vessel. Conclusion The newly formed complex of Medpor-PGA and cells was very similar to testicle in gross view and to normal cartilage in histology. This pilot technique of creating testicular prosthesis by incorporating tissue-engineered cartilage with Medpor demonstrated success.
【摘要】 目的 观察低频超声（20 kHz）辐照联合静脉注射微泡造影剂SonoVue对裸鼠前列腺癌（Du145）移植瘤的抑瘤效应，并探讨其可能的抑瘤机制。 方法 通过细胞移植和瘤块移植方法建立24只裸鼠前列腺癌Du145移植瘤模型，随机分为超声微泡组、单纯超声组、单纯微泡组和对照组，每组各6只。超声微泡组：尾静脉注射0.2 mL SonoVue的同时对瘤体行20 kHz超声辐照，辐照强度200 mW/cm2；单纯超声组：尾静脉注射生理盐水0.2 mL，同时超声辐照2 min；单纯微泡组：尾静脉注射SonoVue 0.2 mL同时行假照，各组均隔天1次，共3次，对照组不做任何处理。治疗后测量瘤体大小，绘制瘤体生长曲线，计算抑瘤率。首次治疗后14 d剥离瘤体，通过光学显微镜、电子显微镜观察肿瘤组织病理改变。免疫组织化学方法观察CD34阳性染色血管，计算肿瘤微血管密度（micro vessel density，MVD），比较各组间MVD的差异。 结果 24只裸鼠均成功植瘤。治疗后超声微泡组瘤体体积均数明显小于其他3组（Plt;0.05），抑瘤率为62.7%。光学显微镜下超声微泡组瘤体组织大部分损伤坏死，电子显微镜下超声微泡组肿瘤内微血管的内皮细胞损伤，线粒体肿大，基底膜断裂。超声微泡组瘤体内CD34阳性染色微血管数减少，其MVD值显著低于其他各组。 结论 20 kHz低频超声辐照联合微泡造影剂SonoVue可有效抑制裸鼠人前列腺癌移植瘤的生长，其抑瘤机制可能是通过超声空化效应破坏肿瘤的微血管实现的。【Abstract】 Objective To investigate the anti-tumor effect induced by low-frequency ultrasound (20 kHz) radiation combined with intravenous injection of microbubbles on human prostate carcinoma xenograft in nude mice, and to discuss its probable mechanism. Methods Human prostate carcinoma xenograft model in 24 nude mice were established with human prostate carcinoma Du145 cells inoculation and sub-graft through mice, which were randomly divided into ultrasound+microbubble, ultrasound, microbubble, and control group, with 6 mice in each group. In the ultrasound+microbubble group, 0.2 mL SonoVue was injected intravenously, followed by 20 kHz ultrasound exposure of 200 mW/cm2 at every other day for 3 times totally. Mice in the ultrasound group and the microbubbles group were only treated with ultrasound radiation and microbubbles injection, respectively. The volume of gross tumors was measured, and tumor growth curve was drawn. The ratio of anti-tumor growth was calculated. The mice were sacrificed 14 days after the last ultrasound exposure. Specimens of the exposed tumor tissues were obtained and observed pathologically under light microscope and transmission electron microscope. CD34 positive vessels were counted in all the tumor slices by immunohistochemistry, and the micro-vessels density（MVD）of the tumor was also calculated. Results Du145 prostate tumor model was successfully established in all the mice. The average gross tumor volume of the ultrasound+microbubble group was significant lower compared with the other two groups after treatment (Plt;0.05), and the ratio of anti-tumor growth was 62.7%. Histological examination showed signs cell injury in the ultrasound+microbubble group. Electron microscopic examination revealed that the endothelium of vessels in the tumor was injured. The amount of CD34 positive vessels and MVD of the ultrasound+microbubble group was less than that of the other two groups. Conclusion The low-frequency ultrasound of 20 kHz exposure combined with microbubbles can be used to ablate human prostate carcinoma xenograft in nude mice, which is probably realized through micro-vessels destroyed by cavitation effect of ultrasound.