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 觀察低頻超聲（20 kHz）輻照聯合靜脈注射微泡造影劑SonoVue對裸鼠前列腺癌（Du145）移植瘤的抑瘤效應，并探討其可能的抑瘤機制。 方法 通過細胞移植和瘤塊移植方法建立24只裸鼠前列腺癌Du145移植瘤模型，隨機分為超聲微泡組、單純超聲組、單純微泡組和對照組，每組各6只。超聲微泡組：尾靜脈注射0.2 mL SonoVue的同時對瘤體行20 kHz超聲輻照，輻照強度200 mW/cm2；單純超聲組：尾靜脈注射生理鹽水0.2 mL，同時超聲輻照2 min；單純微泡組：尾靜脈注射SonoVue 0.2 mL同時行假照，各組均隔天1次，共3次，對照組不做任何處理。治療后測量瘤體大小，繪制瘤體生長曲線，計算抑瘤率。首次治療后14 d剝離瘤體，通過光學顯微鏡、電子顯微鏡觀察腫瘤組織病理改變。免疫組織化學方法觀察CD34陽性染色血管，計算腫瘤微血管密度（micro vessel density，MVD），比較各組間MVD的差異。 結果 24只裸鼠均成功植瘤。治療后超聲微泡組瘤體體積均數明顯小于其他3組（Plt;0.05），抑瘤率為62.7%。光學顯微鏡下超聲微泡組瘤體組織大部分損傷壞死，電子顯微鏡下超聲微泡組腫瘤內微血管的內皮細胞損傷，線粒體腫大，基底膜斷裂。超聲微泡組瘤體內CD34陽性染色微血管數減少，其MVD值顯著低于其他各組。 結論 20 kHz低頻超聲輻照聯合微泡造影劑SonoVue可有效抑制裸鼠人前列腺癌移植瘤的生長，其抑瘤機制可能是通過超聲空化效應破壞腫瘤的微血管實現的。【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.
ObjectiveTo investigate the inhibitory effect of short hairpin RNA (shRNA) mediated contactin-1 (CNTN1) gene silencing on growth of human breast cancer cell line MDA-MB-468 transplanted tumors in nude mice.MethodsEighteen nude mice (4-week-old male BALB/c) were randomly equally divided into three groups: blank control group, empty vector group, and silencing group. The MDA-MB-468 cells (blank control group), MDA-MB-468 cells transfected by nonsense shRNA (empty vector group), and MDA-MB-468 cells transfected by shRNA (silencing group) were collected in the logarithmic growth period, respectively. The subcutaneous tumor models of nude mice were prepared by the subcutaneous injection of the different group cells. The tumor growth was observed and the expressions of CNTN1 and Ki-67 proteins in the transplanted tumor were detected by the immunohistochemistry.ResultsThe xenograft models of human breast cancer cells were established successfully. The tumor growth in the silencing group was significantly slower than that of the other two groups at every 3 d point (P<0.05). The tumor volume and the tumor weight in the silencing group were significantly smaller or slighter than those of the other two groups at day 18 (P<0.05). The positive rates of CNTN1 and Ki-67 protein expressions in the tumor tissues of the silencing group were lower than those of the other two groups (P<0.05), respectively.ConclusionSilencing expression of CNTN1 gene might inhibit growth of breast cancer cell line MDA-MB-468 transplanted tumors in mude mice.
Objective To study effect of carcinoembryonic antigen (CEA) positive targeted lymphocytes on gastric cancer cells in vitro and in vivo. Methods The peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers. The recombinant vector anti-CEA-scFv-CD3ζ-pcDNA3.0 was transfected into the PBMCs by lipofectamine 2000, by this means, the CEA special lymphocytes were obtained. Meanwhile, the PBMCs transfected with empty plasmid pcDNA3.0 were used as control (empty vector lymphocytes). The different lymphocytes and gastric cancer cells (CEA positive KATOⅢ gastric cancer cells and CEA negative BGC-823 gastric cancer cells) were co-cultured, then the ability to identify the gastric cancer cells and it’s effect on apoptosis of gastric cancer cells were observed at 24 h or 36 h later respectively. The CEA special lymphocytes and empty vector lymphocytes were injected by the tail vein of nude mice bearing gastric cancer cells, then it’s effect on the tumor was observed. Results ① The CEA special lymphocytes could strongly identify the KATOⅢ gastric cancer cells (identification rate was 72.3%), which could weakly identify the BGC-823 gastric cancer cells (identification rate was 7.8%). ② The apoptosis rate of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly higher than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (P=0.032), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (P=0.118). ③ The tumor volume of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly smaller than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (F=5.010, P<0.01) or the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells (F=4.982, P<0.01), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (F=1.210, P>0.05). Conclusion CEA special lymphocytes can promote cell apoptosis and inhabit tumor reproduction of CEA positive gastric cancer cells in vitro and in vivo.
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.
ObjectiveTo study the expression of lipid associated with neutrophil gelatinase associated lipocalin (NGAL) in nude mice orthotopic pancreatic cancer tissues and the relationship between the occurred and development of pancreatic cancer. MethodsThe expressions of NGAL mRNA and protein of pancreatic cancer tissues and their adjacent tissues, and normal pancreatic tissues in nude mice were detected by using RT-PCR and immunohistochemical methods. ResultsThe expressions of NGAL mRNA in pancreatic cancer tissues and adjacent tissues were significantly higher than that in normal pancreatic tissues (P < 0.05), and the expression of NGAL mRNA in pancreatic carcinoma tissues was significantly higher than that in para carcinoma tissues (P < 0.05). The strong positive expression rate of NGAL protein in pancreatic carcinoma tissues was significantly higher than thoes in para carcinoma tissues and normal pancreatic tissues (P < 0.05). ConclusionsNGAL is highly expressed in pancreatic cancer tissues, and NGAL may be an important regulatory factor in the development of pancreatic cancer.
ObjectiveTo investigate MUC1 over-expression on chemotherapy of 5-fluorouracil and cisplatin for esophageal cancer cells. MethodsMUC1 over-expression and stable silencing of MUC1 expression esophageal cancer cell lines were constructed. Xenograft model of esophageal cancer was established in nude mice. Cisplatin (8 mg/kg, day 1 and day 7)and 5-fluorouracil (20 mg/kg, day 1 to 6)were injected intraperitoneally. Tumor volume and body weight of nude mice were measured. Tumor growth curve and body weight curve were drawn, and tumor inhibitory rate was calculated. ResultsBoth cisplatin and 5-fluorouracil suppressed tumor growth of MUC1 over-expression esophageal cancer nude mice. Body weight and tumor volume of nude mice of cisplatin and 5-fluorouracil groups were significantly smaller than those of the control group (P < 0.05), and the inhibitory effects of cisplatin were significantly greater than those of 5-fluorouracil (P < 0.05). There was no significant inhibitory effect in stable silencing of MUC1 expression esophageal cancer nude mice. ConclusionBoth cisplatin and paclitaxel can suppress the growth of MUC1 over-expression esophageal cancer, and cisplatin has greater inhibitory effects than 5-fluorouracil in tumor volume and body weight of nude mice.
ObjectiveTo investigate the co-transplantation of C57-green fluorescent protein (GFP) mouse epidermis and dermis cells subcutaneously to induce the hair follicle regeneration. MethodC57-GFP mouse epidermis and dermis were harvested for isolation the mouse epidermis and dermis cells. The morphology of epidermis and dermis mixed cells at ratio of 1:1 of adult mouse, dermis cells of adult mouse, cultured 3rd generation dermis cells were observed by fluorescence microscope. Immunocytochemistry staining was used to detect hair follicle stem cells markers in cultured 3rd generation dermis cells from new born C57-GFP mouse. And then the epidermis and dermis mixed cells of adult mouse (group A), dermis cells of adult mouse (group B), cultured 3rd generation dermis cells of new born mouse (group C), and saline (group D) were transplanted subcutaneously into Balb/c nude mice. The skin surface of nude mice were observed at 4, 5, 6 weeks of transplantation and hair follicle formation were detected at 6 weeks by immunohistochemistry staining. ResultsThe isolated C57-GFP mouse epidermis and dermis cells strongly expressed the GFP under the fluorescence microscope. Immunocytochemistry staining for hair follicle stem cells markers in cultured 3rd generation dermis cells showed strong expression of Vimentin and α-smooth muscle actin, indicating that the cells were dermal sheath cells; some cells expressed CD133, Versican, and cytokeratin 15. After transplanted for 4-6 weeks, the skin became black at the injection site in group A, indicating new hair follicle formation. However, no color change was observed in groups B, C, and D. Immunohistochemical staining showed that new complete hair follicles structures formed in group A. GFP expression could be only observed in the hair follicle dermal sheath and outer root sheath in group B, and it could also be observed in the hair follicle dermal sheath, outer root sheath, dermal papilla cells, and sweat gland in group C. The expression of GFP was negative in group D. ConclusionsCo-transplantation of mouse epidermis and dermis cells can induce the hair follicle regeneration by means of interaction of each other. And transplantation of isolated dermis cells or cultured dermis cells individually only partly involved in the hair follicles formation.
ObjectiveTo investigate the survival rate of core fat tissue with different diameters by advanced fat harvesting instrument. MethodsBased on core fat transfer by 1 mL syringe, the fat harvesting instrument was modified with different diameters, including 4, 6, 8, and 10 mm respectively. Between May 2014 and April 2015, the fat harvesting instrument with diameters of 4, 6, 8, and 10 mm was respectively used to harvest abdominal fat in 3 of 12 patients undergoing autologous fat transplantation. The glucose transportation quantities and the fat cell viability were measured. Then 64 nude mice at the age of 3-4 weeks were randomly divided into 4 groups (groups A, B, C, and D, n=16). And 0.5 mL fat harvested with diameters of 4, 6, 8, and 10 mm was implanted into the dorsal subcutaneous space. After fat transplantation, the mice survival and the appearance at the recipient site were observed. At 1, 2, 4, and 8 weeks after fat transplantation, the grafted fat was harvested for gross, histological and immunohistochemical observations; the intact adipocytes and capillary were counted. ResultsThe glucose transportation quantities gradually increased with increased diameter, showing significant difference among groups (P<0.05). And the fat cell viability had a rising tendency, showing significant differences when comparing groups A and B with group D (P<0.05). With the time passing by, the protuberant appearance became flat at the recipient site, but the appearance of groups C and D was better than groups A and B. Normal shape of the fat and capillary were found in groups C and D. At immediate and 1 week after fat transplantation, there was no significant difference in fat weight among 4 groups (P>0.05); the fat weight of group A was significantly less than that of groups B, C, and D (P<0.05) at 2, 4, and 8 weeks after fat transplantation, and it was significantly less in group B than groups C and D (P<0.05), but no significant difference was found between groups C and D (P>0.05). Histological and immunohistochemical observations showed better integrity of the cells, less necrosis, and higher vascular density in group D than groups A, B, and C as time extension. The adipocyte integrity of group A was significantly worse than that of other 3 groups at other time points (P<0.05) except at 1 week (P>0.05). At each time point, the capillary counting had an increasing trend with increased diameter in all groups, showing significant difference among groups (P<0.05). ConclusionWith diameters within 10 mm, the thicker the core fat is transferred, the better integrlity, higher vessel density, and quicker revascularization time can be predicted. So the postoperative appearance could be maintained longer.
Objective To establish a xenograft model of hydroxycamptothecine (HCPT)-resistant human gastric cancer cell line (SGC-7901/HCPT) in nude mice and study its biological characteristics. Methods The SGC-7901 and SGC-7901/ HCPT cells were cultured in vitro. The cell suspension was injected subcutaneously into the nude mice. When the subcutaneous carcinoma was 1.0 cm in diameter, it was cut off and divided into pieces of 0.1-0.2 cm in diameter． Then the small pieces of tumor were re-transplanted subcutaneously into the second generation nude mice until the fourth generation. The morphological feature, ultramicro-structure, and growth characteristics of the fourth generation transplanted tumor were examined. The drug resistance was measured by methyl thiazolyl tetrazolium (MTT) assay. Results The transplanted tumor in nude mice was round or oval, and many blood vessels were on its surface. Under the light microscope, the sizes of SGC-7901 transplanted tumor cells were similar, and sizes of cell nuclei were also similar; Meanwhile, the morphous of SGC-7901/HCPT transplanted tumor cells were irregular and in disorder, and the size of the cell nuclei was different from each other. Under the electron microscope, the mitochondria and endoplasmic reticulum of SGC-7901 transplanted tumor cells were nearly normal and no swelling in cell nuclei; Meanwhile the cell nuclei of SGC-7901/HCPT transplanted tumor cells were lightly swelled, a the mitochondria and endoplasmic reticulum were obviously swelled. By MTT assay, compared with SGC-7901 transplanted tumor cells, the resistance index of SGC-7901/HCPT transplanted tumor cells was 9.02±0.78 in HCPT, and resistance index to Adriamycin， Mitomycin C, 5-fluorouracil, and Etoposide was 1.24±0.09, 1.31±0.17, 0.96±0.12, and 1.07±0.16, respectively. Conclusions A transplanted tumor model of SGC-7901/HCPT in nude mice is established successfully， and showing stable drug resistance to HCPT and no cross-resistance to other chemotherapeutics, which can be used for further experiments.