Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
【摘要】 目的 探討LIM礦化蛋白（LIM mineralization protein，LMP）-1和LMP-3雙基因共轉染骨髓間充質干細胞（bone mesenchymal stem cells，BMSC）的表達情況。 方法 采用人工設計合成人LMP-1和LMP-3基因片段，分別與質粒pEGFP-N2連接，經酶切、測序鑒定后。分離培養新西蘭兔BMSC，用脂質體包裹轉染BMSC，按轉染情況分為5組:未轉染組（A組）、轉染空載體組（B組）、轉染LMP-1基因組（C組）、轉染LMP-3基因組（D組）、LMP-1與LMP-3雙基因共轉染組（E組）。采用實時聚合酶鏈反應（real-time polymerase chain reaction，RT-PCR）和蛋白質印跡法檢測LMP-1和LMP-3的表達。 結果 酶切及測序表明真核表達質粒pEGFP-N2-LMP-1和pEGFP-N2-LMP-3構建成功。E組可同時較高水平表達LMP-1和LMP-3分子。對RT-PCR及蛋白質印跡法檢測結果行灰度值測量并行統計學分析顯示:LMP-1 mRNA及蛋白水平的表達，5組間差異有統計學意義（Plt;0.05），但E組與C組的差異無統計學意義（Pgt;0.05）；LMP-3 mRNA及蛋白水平的表達，5組間差異有統計學意義（Plt;0.05），且E組與D組差異也有統計學意義（Plt;0.05）。 結論 雙基因共轉染的BMSC能在體外同時表達LMP-1與LMP-3，為基因修復骨缺損帶來新思路。【Abstract】 Objective To study the expression of LIM mineralization protein (LMP)-1 and LMP-3 genes after cotransfecting them into bone mesenchymal stem cells (BMSC) of rabbit in vitro. Methods Fragments of LMP-1 gene and LMP-3 gene were gained through artificial synthesis, and were constructed respectively into the plasmid vector pEGFP-N2. The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes. The plasmids carrying LMP-1 and LMP-3 genes were cotransfected into chondrocytes by liposome method. According to the transfected situation, the BMSC were divided into 5 groups: the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by LMP-1 (Group C), the group transfected by LMP-3 (Group D) and the group transfected by both LMP-1 and LMP-3 (Group E). The expressions of LMP-1 and LMP-3 were detected by RT-PCR and western bloting technique. Results The plasmid pEGFP-N2-LMP-1 and pEGFP-N2-LMP-1 were obtained successfully by cloning technique and verified by nucleotide sequencing and enzymes. The LMP-1 and LMP-3 molecules were both expressed at a high level in Group E. The results of RT-PCR and western bloting were measured with the grey value. For the expression of LMP-1 mRNA and protein of LMP-1, the differences between groups A, B and groups C, D, E were significant (P＜0.05), while the difference between groups C and E was not significant (P＞0.05); For the expression of LMP-3 mRNA and protein of LMP-3, the differences between groups A, B and groups C, D, E were significant (P＜0.05), and the difference between groups D and E was also significant（P＜0.05). Conclusion LMP-1 and LMP-3 genes can be expressed effectively after being cotransfected into BMSC, which provides a basis for gene therapy for treating bone defects.
目的 探討魚膽汁對兔腎臟的影響及其機制。 方法 將實驗新西蘭大耳白兔隨機分為灌胃組（GP組，n=19）與靜脈注射組（VI組，n=15），根據體重分別按3 mL/kg、0.3 mL/kg的劑量通過灌胃或耳緣靜脈注射方式給予魚膽汁。采集魚膽汁處理前與處理后1～5 h的血標本，測定腎功能、酸堿平衡及電解質指標，記錄GP組每個采樣點前20 min尿量及魚膽汁處理前、處理后5 h的尿常規。魚膽汁處理后5 h處死動物取腎做病理學檢查。 結果 給予一定量魚膽汁后5 h內，兩組兔血肌酐（Scr）、尿素氮、K+呈升高趨勢（P均＜0.05），而血HCO3?濃度呈下降趨勢（P＜0.05），其中VI組兔Scr、血K+改變早于GP組。GP組記錄尿量明顯下降，尿pH值升高，蛋白定量試驗、隱血試驗結果均呈陽性。兩組兔腎組織病理檢查均顯示腎小球血管充盈，少量中性粒細胞浸潤；腎小管水腫及間質充血，部分有局灶性出血，腎間質損傷較腎小球更為嚴重。 結論 無論經由消化道還是血管給予實驗兔魚膽汁均可導致急性腎功能損傷，與魚膽汁造成急性腎實質損傷、特別是腎小管間質損傷有關。
Objective To detect the cell density, apoptotic rate, and the expressions of BNIP3 in nucleus pulposus of degenerative intervertebral disc of rabbits, so as to further understand the mechanism of intervertebral disc degeneration. Methods Thirty male New Zealand white rabbits, aging 3 months and weighing (2.3 ± 0.2) kg, were divided into sham operation group (control group, n=10) and intervertebral disc degeneration model group (experimental group, n=20). Interbertebral disc degeneration models were establ ished by puncture of L3,4, L4,5, and L5,6 intervertebral discs in the experimental group; intervertebral discs were exposed only and then sutured in the control group. The degree of intervertebral disc degeneration was evaluated according to Pfirrmann classification by MRI at 4 and 8 weeks after establ ishing models. Apototic cells were determined by TUNEL and histological methods, and the immunohistochemical staining was performed to detect the expressions of BNIP3 in nucleus pulposus of intervertebral disc. Results MRI examination showed that the signal intensity decreased gradually at 4 and 8 weeks in the experimental group. There wassignificant difference in the degree of intervertebral disc degeneration between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The histological observation and TUNEL test showed that high density of nucleus pulposus cells and only a few apoptotic cells were observed in the control group; at 4 and 8 weeks, the density of nucleus pulposus cells decreased gradually with more apoptotic cells in the experimental group. There were significant differences in the nucleus pulposus cell density and positive rate of TUNEL staining between 2 groups, and between at 4 weeks and at 8 weeks in the experimental group (P lt; 0.05). The expression of BNIP3 of nucleus pulposus was negative in the control group; however, in the experimental group, the positive expression rates of BNIP3 of nucleus pulposus (the gray values) were 13.45% ± 1.16% and 32.00% ± 1.82% (194.32 ± 4.65 and 117.54 ± 2.11) at 4 and 8 weeks respectively, showing significant differences (P lt; 0.05). Conclusion The decrease of cell density in nucleus pulposus is involved in the development of intervertebral disc degeneration. Cell apoptosis is one of reasons in the decrease of nucleus pulposus cell; BNIP3 is involved in nucleus pulposus cell apoptosis in the degenerative intervertebral disc.
Objective To observe the effect of cationic liposomal ceftazidime (CLC) combined with nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) in the treatment of chronic osteomyelitis of rabbits. Methods Thirty healthy New Zealand white rabbits (4-6 months old; weighing, 2-3 kg) were selected to prepare the chronic osteomyelitis models. After 4 weeks, the gross observation, X-ray examination, and bacteriological and histopathological examinations were done; the models were made successfully in 27 rabbits. Of 27 rabbits, 24 were randomly divided into 4 groups (n=6): only debridement was performed in group A; ceftazidime was given (90 mg/kg), twice a day for 8 weeks after debridement in group B; ceftazidime and n-HA/β-TC were implanted after debridement in group C; and CLC and n-HA/β-TCP were implanted after debridement in group D. Before and after treatments, X-ray examination was done, and Norden score was recorded. At 8 weeks after treatment, the specimens were harvested for gross observation and for gross bone pathological score (GBPS) using Rissing standard; half of the specimens was used for histological observation and Smeltzer scoring, the other half for bacteriological examination and calculation of the positive rate of bacteria culture. Results At 8 weeks after treatment, Norden score of group D was significantly lower than that of groups A, B, and C (P lt; 0.05), but no significant difference was found among groups A, B, and C (P gt; 0.05). At 8 weeks after treatment, sinus healed in groups C and D, but sinus was observed in groups A and B; the GBPS scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The Smeltzer scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The positive rates of bacteria culture of groups C (0) and D (0) were significantly lower than those of group A (25.0%) and group B (16.7%) (P lt; 0.05). Conclusion CLC combined with n-HA/β-TCP has good effect in treating chronic osteomyelitis of rabbits, and it has better effect in treating chronic osteomyelitis of rabbits than ceftazidime with n-HA/β-TCP.
【Abstract】 Objective To investigate both incidence and mechanism attributing to steroid-associated osteonecrosisof femoral head(ONFH) using an experimental protocol with a single low-dose l i popolysaccharide (LPS) injection andsubsequently three injections of high-dose methylprednisolone (MPS). Methods Twenty-five New Zealand white rabbits with body weight of (3.0 ± 0.3) kg were divided randomly into 2 groups. In treatment group, 19 rabbits received one intravenous injection of LPS (10 μg/kg); 24 hours later, three injections of 20 mg/kg of MPS were given intramuscularly at an interval of 24 hours. Additional 6 rabbits which received normal sal ine injection at the same time point were used as controls(control group). The blood samples were collected for hematological examinations before and after LPS injection, MRI was performed on bilateral hip six weeks after last MPS injection, meanwhile, bone marrow was aspirated from femoral head region to evaluate stem cell’s activity. Bilateral femoral heads were harvested to make histopathology examination. Results All animals survived throughout the experiment period except one death on the second day after LPS injection. In the histopathological examinationfor the femoral head, ONFH+ was observed in 16 rabbits (88.9%), and the lesions were mainly in the metaphysis. In ONFH+ rabbits, micro vessels fibrous thrombosis and extravascular marrow fat cell size increasing were found around necrotic bone; The femoral heads of control group had no changes. MRI accurate ratio was 93.8% (15/16). Compared to basel ine, a significant decrease in ratio of tissue plasminogen activator/plasminogen activator inhibitor 1 and activated partial thromboplatin time, and a significant increase in ratio of low-density l ipoprotein/high-density l ipoprotein were only found in ONFH+ rabbits (P lt; 0.05). Meanwhile there was a significant decrease in the number of CFU-F (8.50 ± 9.63) compared with the control (70.17 ± 7.78, P lt; 0.05). Conclusion A single low-dose LPS injection and subsequent three injections of high-dose MPS is effective on building steroid-associated ONFH model, coagulation and l ipometabol ism abnormal ity, activity degeneration of stem cell may be the key factors of ONFH.
【摘要】 目的 研究不同壓力二氧化碳（CO2）氣腹對糖代謝的影響。 方法 18只雌性健康新西蘭大白兔按CO2氣腹壓力隨機均分為氣腹壓0 mm Hg （1 mm Hg=0.133 kPa）（Ⅰ組）、氣腹壓10 mm Hg（Ⅱ組）和氣腹壓15 mm Hg（Ⅲ組）。每組兔均在不同的壓力下接受氣腹1 h。在CO2氣腹前（T0）、氣腹后30 min （T1）、氣腹后60 min （T2） 測定動脈血氣分析值、血糖（Glu）、胰島素（Ins）和胰高糖素（Gln）。 結果 氣腹后30 min 、60 min，Ⅱ組與Ⅰ組比較，PaCO2、Glu 、Gln增加（Plt;0.05），pH值和Ins下降（Plt;0.05），Ⅲ組各參數變化更為顯著（Plt;0.01）。結論 CO2氣腹后機體可能處于較強烈的應激狀態，導致血糖升高。【Abstract】 Objective To study effects of different intraabdominal pressure of carbon dioxide (CO2) pneumoperitoneum on blood glucose level in rabbits. Methods Eighteen female healthy rabbits weighed 2.1-3.3 kg were randomly divided into three groups equally based on pneumoperitoneum pressure: 0 mm Hg (1 mm Hg=0.133 kPa) group (groupⅠ),10 mm Hg group (groupⅡ) and 15 mm Hg (groupⅢ). Each group received 1h pneumoperitoneum under diffent pressure. Blood samples were taken before CO2 pneumoperitoneum, at 30 and 60 minutes after pneumoperitoneum for the measure-ments of arterial blood gas, blood glucose (Glu), insulin (Ins) and glucone (Gln). Results After pneumoperitoneum at 30 and 60 minutes, compared with groupⅠ, PaCO2,Glu and Gln were significantly raised in groupⅡ(Plt;0.05), pH and Ins were markedly decreased (Plt;0.05). Even more significant changes were observed in group Ⅲ(Plt;0.01). Conclusion After CO2 pneumoperitoneum, body is in a relatively b stress, so blood glucose is decreased.
Fluorescein angiography(FA)was performed in 31 pigmented rebbits.The angiograms were evaluated as prints and as negative film under a light microscope.The patterns of retinal pigment epithelial(RPE)cells were studied by scaning electron microscopy and fluorescein light one,compared with other rabbits belonging to the same species.In 58 eyes,we observed the hexagonal pattern of RPE cell.It showed central hypofluorescent area surrounded by hyperfluorescent rim,which was easily seen away from the medullary rays by three or more disc diameters and became larger in the periphery than that in the posterior pole.There were no finding in four lightly pigmented eyes. （Chin J Ocul Fundus Dis,1994,10:226-228）
Objective To research the effect of porcine acellular dermal matrix in the reconstruction of abdominal wall defects in rabbits, and to investigate the appl ication feasibil ity of xeno-transplantation of acellular dermal matrix. Methods The porcine acellular dermal matrix was prepared from a health white pig. Twenty-six Japanese white rabbits (weighing 2.2-2.3 kg, female or male) were randomly assigned to 2 groups: the control group (n=6) and the experimental group (n=20). In the control group, the full-thickness abdominal wall defect of 5.0 cm × 0.5 cm was made, and the defect wassutured directly; in the experimental group, the full-thickness abdominal wall defect of 5.0 cm × 2.5 cm was made, and the defect was repaired with porcine acellular dermal matrix patch at the same size as the defect. At 5 weeks after surgery, the incidence of hernia and the intra-abdominal adhesions were observed and the wound breaking strength was compared between the patchfascia interface and the fascia-fascia interface. The graft vascularization was evaluated through histological analysis at 6 months after surgery in the experimental group. Results No hernia occurred in all rabbits of 2 groups. At 5 weeks after surgery, heal ing was observed between patch and the muscularfascia; the vascularization was seen in the porcine acellular dermal matrix patch. There was no significant difference in the adhesion grade (Z= —0.798, P=0.425) between the experimental group (grade 2 in 1 rabbit, grade 1 in 5, and grade 0 in 12) and the control group (grade 1 in 1 and grade 0 in 5). No significant difference was found (t= —0.410, P=0.683) in the breaking strength between the patch-fascia interface in the experimental group [(13.0 ± 5.5) N] and the fascia-fascia interface in control group [(13.6 ± 4.0) N]. In the experimental group, the small vessels and the infiltration of inflammatory cells were observed in the porcine acellular dermal matrix patch after 5 weeks through histological observations. The junctions of the patch-fascia interface healed with fibrous connective tissue. At 6 months after surgery, the inflammation was subsided and the collagen fiber of the patch was reconstructed. Conclusion The porcine acellular dermal matrix patchhas good results in repairing full-thickness abdominal wall defect. The patch-fascia interface has siml iar breaking strength to the fascia-fascia interface. The collagen fibers of the patch are reconstructed.