Objective To study the progressive development of the retinas through an observation on the histological changes of the retinas from neonatal mice of different day-ages. Methods The retinas from the mice of 1 to 20 days of age were examined by light microscopy,and from 1 to 3 days,by autoradiography. Results The retinas of the mice below 3 days of age only had the RPE cells layer,the neuroblast layer and the ganglion cell layer.With the increase in dayage,the retinas developed gradually and would be mature in the 20th day. Conclusions The retinas of mice is a kind of immature tissue before the 20th days,so it can be considered as transplantation donors. (Chin J Ocul Fundus Dis, 1999, 15: 174-176)
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.
ObjectiveTo observe the effect of using tungsten drills to prepare mouse knee osteochondral injury model by comparing with the needle modeling method, in order to provide an appropriate animal modeling method for osteochondral injury research.MethodsA total of 75 two-month-old male C57BL/6 mice were randomly divided into 3 groups (n=25). Mice in groups A and B were used to prepare the right knee osteochondral injury models by using needles and tungsten drills, respectively; group C was sham-operation group. The general condition of the mice was observed after operation. The samples were taken at 1 day and 1, 2, 4, and 8 weeks after modeling, and HE staining was performed. The depth, width, and cross-sectional area of the injury site at 1 day in groups A and B were measured, and the percentage of the injury depth to the thickness of the articular cartilage (depth/thickness) was calculated. Toluidine blue staining and immunohistochemical staining for collagen type Ⅱ were performed at 8 weeks, and the International Cartilage Research Society (ICRS) score was used to evaluate the osteochondral healing in groups A and B.ResultsAll mice survived to the completion of the experiment. HE staining showed that group C had normal cartilage morphology. At 1 day after modeling, the injury in group A only broke through the cartilage layer and reached the subchondral bone without entering the bone marrow cavity; the injury in group B reached the bone marrow cavity. The depth, width, cross-sectional area, and depth/thickness of the injury in group A were significantly lower than those in group B (P<0.05). At 1, 2, 4, and 8 weeks after modeling, there was no obvious tissue filling in the injured part of group A, and no toluidine blue staining and expression of collagen type Ⅱ were observed at 8 weeks; while the injured part of group B was gradually filled with tissue, the toluidine blue staining and the expression of collagen type Ⅱ were seen at 8 weeks. At 8 weeks, the ICRS score of group A was 8.2±1.3, which was lower than that of group B (13.6±0.9), showing significant difference (t=−7.637, P=0.000).ConclusionThe tungsten drills can break through the subchondral bone layer and enter the bone marrow cavity, and the injury can heal spontaneously. Compared with the needle modeling method, it is a better method for modeling knee osteochondral injury in mice.
Objective To investigate the mechanism of AMP-activated protein kinase (AMPK) in hepatic ischemia-reperfusion injury (HIRI). Methods ① Grouping. Forty-two mice were randomly divided into Sham group, 4 ischemia reperfusion (IR) group of different times (2, 6, 12, and 24 h), Compound C group, and Compound C+repamycin (Rapa) group, each group enrolled in 6 mice. Compound C group: mice were modeled at 1 h after intraperitoneal injection of Compound C (25 mg/kg). Compound C+Rapa group: mice were modeled at 1 h after intraperitoneal injection of rapamycin (1 mg/kg) and Compound C (25 mg/kg). Mice of 4 IR groups, Compound C group, and Compound C+Rapa group were used to prepare HIRI model. Mice of Sham group were treated only for laparotomy, freeing the first portal hepatis and closing peritoneal. ② To filter the best IR time. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in Sham group and IR groups of 4 different reperfusion time points were measured. The pathological changes of liver tissues were observed by HE staining, and the expressions of related proteins in liver tissue of mice were detected by Western blot. Considering the results of blood biochemical test, HE staining, and Western blot together to determine the best IR point. ③ The exploration of signal pathway for AMPK. The expressions of proliferating cell nuclear antigen (PCNA) were observed by immunohistochemical staining in the liver tissues of IR-12 h group, Compound C group (12 h after IR) and compound C+Rapa group (12 h after IR). The mitochondrial damage was observed by rhodamine 123 staining, and the apoptotic status of liver cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL). Results ① The 12 h after IR was the best observation time point. Compared with IR-12 h group, the levels of ALT and AST in Sham group, IR-2, 6, and 24 h groups were lower (P<0.05). HE staining showed that liver tissue destruction in IR-12 h group was the most severe. Western blot showed that, expressions of AMPKα, phosphorylated adenylate activated protein kinase α (p-AMPKα), Nip3-like protein X (Nix), BCL-2 homologous water-soluble protein (Bax), as well as ratio of autophagy microtubule-associated protein light chain 3 (LC3)Ⅱto LC3Ⅰof Sham group, IR-2, 6, and 24 h group were all lower than those of IR-12 h group (P<0.05), but the expressions of phosphorylated mammalian target of Rapa (p-mTOR) of Sham group, IR-2, 6, and 24 h group were all higher (P<0.05). Therefore, 12 h after IR was the best time to observe. ② Compared with IR-12 h group, the expression level of PCNA protein in liver tissue of Compound C group was lower (P<0.05), the mitochondrial luminescence intensity was weaker and the apoptotic cells were more. Compared with Compound C group, the expression of PCNA protein in the liver tissue of the Compound C+Rapa group was higher (P<0.05), the mitochondrial intensity was stronger and the apoptotic cells were less. ③ Compared with IR-12 h group, the expressions of Nix and p-AMPKα, and ratio of LC3Ⅱ to LC3Ⅰ in liver tissue of Compound C group decreased (P<0.05), while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 increased (P<0.05). Compared with Compound C group, the expressions of p-AMPKα and Nix in the liver tissue of Compound C+Rapa group increased (P<0.05), while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 decreased (P<0.05). Conclusion During the HIRI in mouse, AMPK regulates mitophagy and apoptosis through the mTOR/Nix pathway.
Objective To investigate the role of calcium- and integrin-binding protein-1（CIB1） in oxidized lowdensity lipoprotein（OX-LDL） inhibiting migration of mouse macrophages. Methods To silence CIB1 express of mouse macrophages by RNA interference, then incubating mouse macrophages with OX-LDL, cell migration and cell spreading of mouse macrophages were analyzed. Results At 24-72h after macrophages transfected CIB1 siRNA, the express of CIB1 protein was restrained obviously. To silence CIB1 express could increase migration and spreading of mouse macrophages significantly. Conclusions CIB1 plays the important role in intracellular modulating mechanism of OX-LDL inhibiting mouse macrophages migration.
Objective To construct specifically expressed vascular endothelial growth factor (VEGF)165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA3.1+-VEGF165 to form recombinant plasmid pcDNA3.1+-rho-VEGF165. The correct recombinant plasmid pcDNA3.1+-rho-VEGF165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmidpcDNA3.1+-rho-VEGF165 than that in plasmidpcDNA3.1+-rho-VEGF165 ; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA3.1+-rho-VEGF165 and plasmid pcDNA3.1+-rho-VEGF165 was found. Conclusions The construction of pcDNA3.1+-rho-VEGF165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina. (Chin J Ocul Fundus Dis, 2005,21:106-108)
Objective To investigate the effect of myoblast transplantation on duchenne muscular dystrophy (DMD) and to explore the method and feasibil ity of applying gene therapy to DMD. Methods Myoblast of C57/BL10 mice were cultured using multiple-step enzyme digestion method and differential velocity adherent technique. The morphology of the cells was observed with inverted phase contrast microscope. The cells at passage 4 were labeled with 5-BrdU. Twenty-four DMDmodel mice (mdx mice: aged 4-6 weeks, male, 13.8-24.6 g) were randomly divided into two groups (n=12 per group): group A, 1 × 106/mL labeled myoblast were injected via ven caudal is twice at an interval of 2 weeks; group B: 1 mL DMEM/F12 was injected in the same manner serving as a control group. The mice were killed 4 weeks after operation and the motor abil ity of the mice was detected by one-time exhaustive swimming before their death. HE staining and immunohistochemistry staining observation for 5-BrdU, desmin, and dystrophin (Dys) were preformed, and the imaging analysis was conducted. Results The primary myoblast could be sub-cultured 5-7 days after culture, providing stable passage and sufficient cells. The time of onetime exhaustive swimming was (60.72 ± 5.76) minutes in group A and (47.77 ± 5.40) minutes in group B, there was significant significance between two groups (P lt; 0.01). At 4 weeks after injection, HE staining showed that in group A, there were round and transparent-stained myocytes and the percentage of centrally nucleated fibers (CNF) was 67%; while in group B, there were uneven muscle fiber with such pathological changes as hypertrophia, atrophia, degeneration, and necrosis, and the percentage of CNF was above 80%. Immunohistochemistry staining revealed that the expression of 5-BrdU, desmin, and Dys was positive in group A; while in group B, those expressions were l ittle or negative. Image analysis result displayed that integral absorbency (IA) value of desmin was 489.70 ± 451.83 in group A and 71.15 ± 61.14 in group B (P lt; 0.05) and the ratio of positive area to thetotal vision area was 0.314 3 ± 0.197 3 in group A and 0.102 8 ± 0.062 8 in group B (P lt; 0.05); the Dys IA value was 5 424.64 ± 2 658.01 in group A and 902.12 ± 593.51 in group B (P gt; 0.05) and the ratio of positive area to the total vision area was 0.323 7 ± 0.117 7 in group A and 0.035 2 ± 0.032 9 in group B (P lt; 0.05). Conclusion Myoblast transplantation has certain therapeutic effect on DMD of mice.
Objective To study the effect of transforming growth factor β1(TGF-β1) and insulin-like growth factor 1(IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. Methods Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditionsto induce themto differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes.TGF-β1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups(TGF-β1+IGF-1 group,10 ng/mland 50 ng/ml respectively;TGF-β1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group(without growth factor). In TGF-β1+IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect ofTGF-β1 and IGF-1 on the expression of collagen Ⅱgene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressionsof collagen Ⅱ were compared between two culture patterns. Results In TGF-β1+IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen Ⅱ at 14 days. The gel electrophoresis results showed that the fragment of collagen Ⅱ gene was seen in TGF-β1+IGF-1 group andTGF-β1 group and that no fragment ofcollagen Ⅱ gene was seen in IGF-1 group and control group. The expression of collagen Ⅱ gene was ber in TGF-β1+ IGF-1 group than inTGF-β1 group, showing significant difference(Plt;0.05). Cells expressed more collagen Ⅱ under pellet culture than under monolayer culture. Conclusion IGF-1 could enhance the effect ofTGF-β1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.
【Abstract】 Objective To investigate the role of myosin l ight chain (Myl) in myogenesis in vitro. Methods The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb prol iferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the abil ity of prol iferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was indentified as the control group. Results The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P lt; 0.01), and the latter two did not show any significant difference (P gt; 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The prol iferation assay showed that the eMb prol iferated faster as compared with dMb and gMb (P lt; 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 ± 24.5)/ field, which was significantly larger than that of myotubes from dMb [(47.6 ± 15.5) / field ] and gMb [(39.8 ± 5.1) field ] (P lt; 0.01). There was not statistically significant difference between the latter two groups (P gt; 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P lt; 0.05), and the dose-dependent way was performed.The numbers of myotubes from dMb at 16 hours were (48.2 ± 7.1)/ well in the experimental group and (23.4 ± 4.9)/ well in the control group, and at 6 days were (40.6 ± 10.2)/ field in the experimental group and (63.1 ± 6.1)/ field in the control group.There was statistically significant difference between the experimental and control groups (P lt; 0.01). Conclusion Myl may play a role in myogenesis through the negative effect on the myoblast prol iferation.