Objective To research the biological feature of intervertebral disc nucleus pulposus cells (NPCs) by observing cell morphous, phenotype and ultramicrostructure. Methods The NPCs from 2-week-old healthy rabbit werecultured in DMEM/F12 medium with 15% FBS. The cell biological features were observed by inverted phase contrast microscope, l ight microscope, electron microscope, cell vital ity assay, cell growth curve and cells staining after harvest and during the periods of culturing the primary, the 1st passage and 2nd passage. Results The results of inverted phase contrast microscope showed that the primary passage adhered at 5 days, grew exponentially at 6-8 days, and were subcultured after covering the bottom at 17 days. The phenotype of the NPCs changed from polygon to long fusiform with passage increased; the vital ity assay showed that there was about 95%-97%, 98%-100%, 100% and 75%-80% NPCs survived just after isolation from intervertebral disc, during the period of culturing the primary, the 1st passage and the 2nd passage, respectively. The toluidine blue staining of the NPCs was bly positive, and HE staining showed clear cell nucleus and cytoplasm. The I collagen immunohistochemical staining showed negative results in the 1st passage, but II collagen immunohistochemical staining and safranin O staining showed positive results. However, the I collagen immunohistochemical staining showed positive result in the 2nd passage, and II collagen immunohistochemical staining and safranin O staining showed weakly positive results. The cell growth curve showed the same as the growth course of cell cultured in vitro. The results of TEM showed that there were many glycogen particles and less chondriosomes in the primary passage. With the increased passage, the glycogen particles decreased and the chondriosomes increased, and cell organ became swell. Conclusion This study clarifies the biological feature of NPCs in vitro, providing the experimental basis for the seed cell research of the nuclues pulposus tissue.
Objective To investigate the effect of hepatocyte-l ike cells induced by CD34+ cells in vitro on the repair of the injured hepatic tissues of mice in vivo. Methods Mononuclear cells were isolated from umbil ical blood by density gradient centrifugation and enriched CD34+ cells were obtained. The cells were (1 × 105 cells/mL) cultured in serumfreemedium containing stem cell factor (SCF), hepatocyte growth factor (HGF), EGF, oncostatin M (OSM), bFGF (the concentration were 50, 20, 20, 10, 10 ng/mL respectively) in vitro for 10 days. Forty-eight 6-week-old female ICR mice werechosen to prepare l iver injury model by injecting carbon tetrachloride and 2-acetylamionoflu-orene. The mice were randomly divided into two groups (n=24 per group): the experimental group, the cultured cells were injected into the mice through the tail vein; the control group, the equivalent serum-free medium was injected. Six mice from each group were killed at 7, 14, 21, and 28 days after operation to receive HE staining, PCR gel electrophoresis, immunohistochemistry staining, and hepatic function detection. Results HE staining: the morphology of injured hepatic tissues in the control group recovered to normal 28 days after operation, while in the experimental group, it recovered to normal 14 days after operation. PCR gel electrophoresis and immunohistochemistry staining: the cells expressing human serum albumin were detected in the hepatic tissue of the experimental group at each time point after operation; while in the control group, no such cells were detected within 28 days after operation. Hepatic function detection: the activity of alanine aminitransperase in the control group recovered to normal 14 days after operation; the mean activity of aspartate aminotransferase of two groups failed to recover within 28 days. Conclusion The hepatocyte-l ike cells induced by CD34+ cells in vitro can promote the morphological and functional recovery of the injured hepatic tissue in mice. Moreover, it can be transformed into human-derived hepatic cells in l iver-injured mice.
OBJECTIVE: To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro. METHODS: The skeletal muscle samples were obtained from 20 to 25-week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. RESULTS: The isolated myoblasts were spherical in cell suspension and spindle-like after attached to culture dishes. The myosin specialized immunohistochemical staining was bly positive. A large quantity of skeletal muscle specialized creatine kinase (CK-MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. CONCLUSION: A large number of myoblasts can be available with digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection. The cultured myoblasts have good ability of proliferation and differentiation.
ObjectiveTo analyze the correlations between the immune function and inflammatory factors levels of patients with hepatocellular carcinoma (HCC) and the results of in vitro high-throughput drug sensitivity, and to provide a reference for personalized drug selection for patients with HCC. MethodsThe patients with HCC who met the inclusion criteria from December 2019 to June 2021 in the First Affiliated Hospital of Chongqing Medical University were included. The HCC cells were used to perform in vitro high-throughput drug sensitivity screening, the result was classified into sensitive and insensitive. The correlations between drug sensitivity results and immune function and inflammatory factors levels of corresponding patients were analyzed, and the relation between these indexes (P<0.05) and drug sensitivity of HCC cells to drugs or combination regimen of drugs was further analyzed by univariate logistic regression. ResultsA total of 74 patients with HCC were included in this study. The results showed that the level of interleukin-6 was negatively correlated with sorafenib, caffezomib, gemcitabine, oxaliplatin + epirubicin + irinotecan + 5-fluorouracil, oxaliplatin + irinotecan + epirubicin, and oxaliplatin + epirubicin regimens on the inhibition rates of HCC in vitro (P<0.05), and positively correlated with bortezomib (P<0.05). However, the level of interleukin-6 was not related to the sensitivity of HCC cells to these single drugs or combined regimens (P>0.05). Meanwhile it was found that tumor necrosis factor (TNF)-α was negatively correlated with cabotinib, apatinib, caffezomib, and epirubicin on the inhibition rates of HCC in vitro (P<0.05), and positively correlated with epirubicin (P<0.05). But only it was found that tumor necrosis factor-α level was related to the sensitivity of HCC cells to epirubicin (P<0.05). ConclusionsTumor necrosis factor-α level in peripheral blood of patients with HCC has a certain relation with epirubicin on inhibition rate of HCC in vitro and it might have a certain value in predicting sensitivity of HCC cells to epirubicin. Meanwhile, although it is found that level of IL-6 is related to sorafenib, caffezomib, gemcitabine, or including combination regiems including oxaliplatin and epirubicin on inhibition rates of HCC in vitro, their value is not found in predicting sensitivity of HCC cells to these single drugs or combined regimens.
Objective To understand the effect of estradiol in different concentrations on proliferation of diverse mammary primary cells in vitro. Methods The primary cells of cancer tissue, the adjacent tissue to tumors and normal mammary tissue from patiens with breast cancer were obtained using collagenase digesting method. All the tissue samples were cultivated in vitro, and were given estradiol in different concentrations. The effect of estradiol on the proliferation of those primary cells was measured by MTT. Results Estradiol remarkedly promoted the proliferation of primary cells of cancer tissue and peritumor tissue in vitro, whose ER expression were positive. Whereas, the promotion effect of estradiol on the proliferation of normal mammary primary cells was relatively weak, and there was no correlation between the promotion effect with the expression of ER in cancer tissue. Conclusion The risks of occurrence and relapse of breast cancer would increase significantly when the concentration of estradiol is no less than 103 pmol/L in vivo.
Objective To investigate the feasibility oftissue engineered intervertebral disc for regeneration of discs. Methods A three-dimensional porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold was fabricated by temperature induced phase separation method. Human fetal disc cells were isolated and cultured in vitro. The disc cells labeledwith a PKH-26 fluorescent dye were seeded into a threedimensional porous scaffold. The proliferation of disc cells with PKH-26 fluorescent labels was assessed by using MTT uptake, laser fluorescence microscopy and SEM. Results Human fetal disc cells displayed a polygonal shape in primary monolayer culture. A regular arrangement and microtubules orientationstructure scaffold with 50-300 μm in diameter was fabricated by thermal-induced phase separation technique. MTT uptake and fluorescent microscopy examination indicated that the seeded disc cells were viable and showed proliferation activity within a porous scaffold. Conclusion The above findings support potential applications of tissue engineered disc in treatment of disc degenerative diseases.
Objective To study the degradable properties of 3D-SC artificial skin in vitro. Methods The 3D-SC artificial skin materials wererespectively immersed into the solutions of 0.9% normal saline (control group), pancreatic tissue liquid (experimental group 1), physiological buffer (Hanks balanced salt solution，experimental group 2) and 0.2 mol/L phosphate buffer (pH 7.4，experimentalgroup 3), and the degradation was carried out at 37℃. The quality lost ratioswere determined on the 3rd day, the 5th day, the 7th day, the 9th day, 11th dayand 14th day in the experimental group 1, while on the 3rd day, 7th day, 14th day, 15th day, 21st day and 30th day in the other groups. Results The 3D-SC artificial skin was degraded completely in pancreatic tissue liquid about within 14 days in the experimental group 1; in the control group, and in the experimental groups 2 and 3, the degradation ratios were 868%±2.30%,28.51%±10.68% and 7.35%±0.61% on the 14th day; 71.83%±2.58%, 91.32%±1.87% and 75.64%±6.13% on the 15th day, being significant difference between the control group and the experimental group 2(Plt;0.01); and 91.87%±8.15%, 95.62%±1.36% and 92.10%±2.26% on the 30th day, being no significant differences between these 3 groups(Pgt;0.05), respectivelies. Conclusion The 3D-SC artificial skin materials have good degradable properties. The trend of degradation speed is from slow to quick and then to slow without enzyme.
【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.
Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.
