Objective To investigate the serum levels of endostatin and vascular endothelial growth factor ( VEGF) at different therapy stages of mouse Lewis lung carcinoma, and elucidate the relation to the progress and prognosis of tumor. Methods Forty-four Lewis lung carcinoma-bearing C57BL/6 mice were randomly divided into 4 groups, ie. a non-therapy group, a chemotherapy group, a gene therapy group, and a combination therapy group ( chemotherapy plus gene therapy) . Eleven healthy mice were included as normal control group. Serum was collected on the 0th, 5th, 19th day after therapy for measurement of endostatin and VEGF by ELISA. The correlations were analysed between endostatin and VEGF levels in each group. Results ( 1) The serum endostatin levels had no significant difference in all groups on the 0th day,but increased significantly on the 5th day in the gene and combination therapy groups than those in other three groups ( all P lt;0. 01) . Then the endostatin level decreased on the 19th day in the gene and combination therapy groups, but still higher than those in the chemotherapy group and the normal group. ( 2 ) On the contrary, serum VEGF levels of the gene and combination therapy groups decreased significantly on the 5th day and increased little on the 19th day, which were both significant lower than those in chemotherapy group on the 5th and 19th day( all P lt; 0. 05) . There were significant diferences between the three therapy groups and the non-therapy group( all P lt;0. 05) . ( 3) Negative correlations between VEGF and endostatin levels were revealed in the gene and combination therapy groups ( r = - 0. 77 and - 0. 761 respectively) .Correlation was not found in the non-therapy and chemotherapy groups. Conclusion The serum levels of endostatin and VEGF might be used as monitoring indices of antiangiogenesis therapy.
ObjectiveTo investigate the effects of over expression of Mash-1 gene on the differentiation of embryonic stem cells (ESC) into neural cells in vitro. MethodsThe ESC of rats (CE3 cells) were transfected with MSCVMash- 1 (MSCV-Mash-1-CE3 group) or MSCV (MSCV-CE3 group). The expression of Mash-1 gene was detected by RT-PCR. After transfection, hanging-drop culture was used to form embryonic bodies, and then embryonic bodies were cultured with neural induction medium. The cell morphology was observed under inverted phase contrast microscopy at 7 and 21 days; the positive rates of neural stem cells marker protein (nestin) and neuron marker protein (β-tubulin Ⅲ) were measured by immunofluorescence staining after cell attachment; and the gene expressions of α-fetal protein (AFP), Brachyury, fibroblast growth factor 5 (FGF-5), Oct3/4, nestin, and β-tubulin Ⅲ were detected by real-time fluorescence quantitative PCR at 0, 1, 7, 14, and 21 days after culture. The CE3 cells were used as control (CE3 group). ResultsCompared with MSCV-CE3 and CE3 groups, the expression of Mash-1 gene in MSCV-Mash-1-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-1-CE3 group had axons growth and showed neural stem cell-like and neuron cell-like morphology (unipolar, bipolar, and multipolar neurons), but few cells had axons growth in MSCV-CE3 and CE3 groups. The positive rates of nestin at 7 days and β-tubulin Ⅲ at 21 days in MSCV-Mash-1-CE3 group were significantly higher than those in MSCV-CE3 and CE3 groups (P<0.05). Real-time fluorescence quatitative PCR results showed that the gene expression of Brachyury was significantly decreased after 1 day (P<0.05), and the gene expressions of FGF-5 and nestin were significantly increased after 1 day (P<0.05) in MSCV-Mash- 1-CE3 group when compared with CE3 and MSCV-CE3 groups; the gene expression of β-tubulin Ⅲ was significantly increased after 7 days (P<0.05). There was no significant difference in above indexes between CE3 and MSCV-CE3 groups (P>0.05). The expressions of AFP and Oct3/4 showed no significant difference among groups at each time point (P>0.05). ConclusionOver expression of Mash-1 gene can promote differentiation of ESC into neural cells in vitro.
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.
Objective To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves. Methods The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis. Results At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B. Conclusion BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.
Allogeneic mouse model of peritoneal heart transplant is a microscopic surgery on small animal with complex techniques. For a beginner, a learning curve of this surgical technique has to be experienced. The learning curve contains three stages:(1) to be familiar with the local anatomy of either donor or recipient mouse; (2) to be capable of collecting donor heart and well preparing the major peritoneal vessels of recipient; (3) to be skillful in the anastomosis of major vessels. The bottleneck of the learning curve is the valid skill of vascular anastomosis. The stepwise essentials are to "understand, be familiar, be accurate, and be quick" in the learning curve.
【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.
ObjectiveTo compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition. MethodsBMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α(HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days. ResultsThe BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P < 0.05). At each time point, compared with group A, the ALP expression, HIF-1αprotein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P < 0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P < 0.05); compared with group C, the HIF-1αprotein relative expression was significantly up-regulated in group B (P < 0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C. ConclusionHypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.
ObjectiveTo study the possbility of using intranasal instillation of fine particulate matter (PM2.5) combined with inhalation of ozone (O3) to establish mouse model of combined pulmonary fibrosis and emphysema (CPFE), and to provide a reference for the establishment of CPFE model.MethodsMale C57/BL6 mice were divided randomly into phosphate buffer saline (PBS) intranasal instillation+air inhalation group, PBS intranasal instillation+O3 inhalation group and PM2.5 intranasal instillation+O3 inhalation group, with 8 mice in each group. The mice were intranasally instilled with PBS or PM2.5 suspension (7.8 mg/kg) followed by air or ozone inhalation 24 hours later, twice a week over 8 weeks. Lung function, bronchoalveolar lavage fluid (BALF) cell counts and classification were detected, the pathological changes of lung tissues in hematoxylin-eosin staining were observed, including inflammation scores and mean linear intercept (Lm). The thickness of collagen deposition in subepithelium was measured in lung tissues in Masson staining, and simultaneously hydroxyproline contents in lung tissues were determined.ResultsCompared to PBS instillation+air inhalation group, inspiratory capacity (IC), total lung capacity (TLC) and chord compliance (Cchord) were increased, FEV25 (the forced expiratory volume at 25 ms)/FVC (forced vital capacity) was decreased, total cell counts in BALF, Lm and lung inflammatory scores were increased, the thickness of the subepithelial collagen layer (SEc/Pbm) or hydroxyproline contents was not changed in PBS instillation +O3 inhalation group; IC was decreased, functional residual capacity (FRC) was increased, TLC was increased, Cchord was decreased, FEV25/FVC and FEV50 (the forced expiratory volume at 50 ms)/FVC were decreased, total cell counts in BALF, Lm, lung inflammatory scores, SEc/Pbm and hydroxyproline contents were increased in PM2.5 instillation+O3 inhalation group. Compared to PBS instillation+O3 inhalation group, IC was decreased, FRC was increased, Cchord was decreased, FEV25/FVC and FEV50/FVC were decreased, total cell counts in BALF, Lm, lung inflammatory scores, SEc/Pbm and hydroxyproline contents were increased in PM2.5 instillation +O3 inhalation group.ConclusionCPFE mouse model can be successfully established by PM2.5 intranasal instillation combined with ozone inhalation for consecutive 8 weeks.
Objective To investigate the effects of hypertonic saline (HTS) treatment on the function and susceptibility to sepsis of reticuloendothelial system (RES) in mice with hemorrhagic shock. Methods Forty percent of total blood volume of male Balb/c mice was withdrawn by cardiac puncture. Two hours later, the mice were treated with blood infusion and normal saline (10 ml/kg) or 7.5% NaCl (10 ml/kg).The survival rate of the mice was observed after cecal ligation and puncture (CLP). The phagocytosis function of the RES was measured by carbon clearance rate(α) and carbon amount ingested by the macrophages of liver and spleen. In vitro, the peritoneal phagocyte function in solutions of different osmotic pressor was measured by assaying neutral red amount taken in. Results The survival rate after CLP in HTS treated group was 70%, whereas all the mice in the normal saline group died. At the third hour after hemorrhagic shock, the RES carbon clearance rate(α) and carbon amount ingested by the macrophages of liver in the HTS treated mice were 5.61±0.42 and 0.59±0.19 respectively, significantly higher than those in the normal saline treated mice (4.15±0.62， 0.42±0.16). In vitro, hyperosmolarity below 40 mmol/L had no significant effects on the phagocytosis activity of peritoneal macrophages in mice. Conclusion Treating hemorrhagic shock with HTS can decrease the susceptibility to sepsis and improve the RES phagocytosis function indirectly.
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.