To investigate the effect of hepatocyte growth factor (HGF) on prol iferation of cultured human eccrine sweat gland epithel ial cells (hESGc) and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the prol iferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 103 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGcwere cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF incontrol group, and in KSFM without HGF and no hESGc in blank group. The cell prol iferation was observed in xperimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the prol iferation of hESGc (P lt; 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell prol iferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.074 3 at 4 days; showing significant differences when compared with control group (P lt; 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell prol iferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.231 0 ± 0.056 7 at 4 days; showing significant differences when compared with control group (P lt; 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation (P lt; 0.01). Conclusion HGF could induce the prol iferation of hESGc and activate the phosphorylation of ERK1/2 protein.
【Abstract】 Objective To reconstruct three different kinds of tympanic membrane in vitro by tissue engineeringtechnique, and to examine their histological structures and mechanical properties. Methods The skin and dura of pig(weight 30 kg) were processed with high satuated sal ine and enzymes to make extracellular matrix. Meanwhile, fibroblasts(1×106 /mL,0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties. Results Fibroblasts cultured were spindle–shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibil ity. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 μm respectively. The tension of the collagen was (1.417±0.030) N/mm2, of the acellular dermal matrix was (24.500±2.040) N/mm2(being close to the tension of normal tympanic membrane, 26.700 N/ mm2),of the acellular dura was (53.300±2.600) N/mm2. Conclusion The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.
In order to study the biological characteristics of tenocyte and fibroblast, the former was obtained from rabbit’s tendon, and the latter from rabbits’s skin. Both cells were cultured according Heuderson’s method. The cell morphology, strapping and expanding time, and the type of collagen fiber synthesized in culture were observed. The results showed that the strapping and expanding time of fibroblast was faster than that of tenocyte. The cellular arrangement of fibroblast was irregular, but that in tenocyte was regular. Type I and III collagen of fibers were found in cultured fibroblost while only type I collagen fibers were found in culture of tenocyte. The tenocyte and fibroblast could be identified individually by strapping and expanding time, arrangement of cells and type of collagen fiber synthesized.
OBJECTIVE This paper was to study the biological characteristics of the transformed human embryonic tendon cells, the relation between cell growth and culture conditions, and to compare these features with that of human embryonic tendon cells. METHODS The pts A58H plasmid had successfully used to transform a tendon cell line from human embryo in our past work. The human embryonic tendon cells and the transformed human embryonic tendon cells were cultured in vitro. In different culture conditions, the growth curve were drawn respectively. Population dependence and proliferation capability of the cells were investigated through plate cloning test and soft agar culture. The collagen secreted by cells was identified by immunohistochemical method. RESULTS In routine culture condition, the growth properties of the human embryonic tendon cell and transformed cells were almost identical. The growth properties of the transformed cells were not changed when the cells were frozen storage. There were changes of growth characteristics of the transformed cells when the culture temperature was changed. The transformed cells could subcultured continually and permanently. The proliferation capability of the transformed cells were ber than that of the human embryonic tendon cells. Moreover, the growth of the transformed cells was serum-dependent, and the phenomenon of contact inhibition was observed. The transformed cells were not able to grow on soft agar culture. They had the capacity of secreting collagen type I. CONCLUSION The transformed human embryonic tendon cells could be subcultured continually and permanently, and their growth could be controlled by changing their culture conditions and they had no malignant tendency in biological characteristics. They could be taken as an ideal experimental material for tendon engineering.
Objective To research the anti-fungal spectrum and activity of the cream containing 1% naftifine-0.25% ketoconazole compared with other two creams that contain of 2% ketoconazole and of 1% terbinafine, respectively. Methods The agar diffusion method was used to judge drug sensitivity. Twenty-nine isolates of pathogenic fungi belonging to 11 species from clinic and three species of Malassezia standard stains were enrolled into the experiment. Organism suspension of each species was spread on the surface of the plate of the optimal media containing 2% agar. Then wells were made in the plate and three types of cream were put in each well respectively. After seven-day incubation, the diameter of the inhibition zone around the well full of each cream was observed and recorded. Results The inhibition zone around the well full of 1% naftifine-0.25% ketoconazole cream for all experiment isolates (Dermatophytes, Candida spp., Sporothrix schenkii, Fonsecaea pedrosoi, Fusarium graminearum, Malassezia furfur, M. globosa and M. sympodialis) was observed, with the mean diameter of 45.46mm. Similarly, the mean diameter of inhibition zone of 2% ketoconazole cream for all experiment isolates was 23.92mm. About 1% terbinafine cream, the mean diameter was 29.81mm but there was no inhibition zone observed around Candida krusei and Candida albicans mycelial-form. There were significant significances for mean diameters of the inhibition zone when comparing 1% naftifine-0.25% ketoconazole cream with 2% ketoconazole cream (P=0.000) and with 1% terbinafine cream (P=0.000). Conclusion The anti-fungal spectrum of 1% naftifine-0.25% ketoconazole cream is wider than that of 1% terbinafine cream. The antifungal activity of 1% naftifine-0.25% ketoconazole cream is ber than that of 2% ketoconazole cream and 1% terbinafine cream.
【Abstract】 Objective To investigate the effect of IGF-1 on the growth of primary human embryonicmyoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the abil ity of prol iferation of myoblasts.The count per minute (CPM) values of myoblasts at different concentrations(1, 2, 4, 8, 16 and 32 ng/mL) of IGF-1 were measured,and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the prol iferation. Then theexperimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growth medium, the controlgroup just received the growth medium. The flow cytometry was used to detect the cell cycle . The method of incorporation of3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts with different concentrations(0, 5,10, 15, 20, 25 and 30 ng/ mL) of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentrationof IGF-1 in stimulating differentiation. Fusion medium with optional concentration of IGF-1 was used in experimentalgroup, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase(CK) weredetected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating prol iferation . It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hoursrespectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, which was in agreementwith the results of the flow cytometry. The optional concentration stimulating prol iferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the prol iferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.
To study the effect of autogeneic PRP on prol iferation and osteogenetic differentiation of human adipose-derived stem cells (ADSCs) in vitro. Methods ADSCs were isolated from adipose tissue obtained from donor undergoing l iposuction and were cultured, and growth condition of the cells was observed by inverted microscope. ADSCs at passage 3 were cultured in adipogenic or chondrogenic medium and underwent identification, immunofluorescence staining observations for CD29 and CD44 were performed. ADSCs at passage 3 were divided into 2 groups: PRP group cultured by osteogenic induction culture medium containing 10 mL/L PRP, and control group cultured by osteogenic induction culture medium without PRP. Then growth condition of the cells was observed by inverted microscope. MTT method was used to observe cell prol iferation activity 1, 2, 3, 4 and 5 days after culture. ALP activity detection was conducted 7, 14, 21 and 28 days after culture. ALP staining was performed on PRP group 7 and 14 days after culture. Al izarin red staining was performed on PRP group 14 days after culture to detect the formation of calcium nodule. Results Under the inverted microscope, most ADSCs at passage 3 were spindle-shaped and the doubl ing time was about 35 hours. Adipogenic and chondrogenic differentiation were confirmed, and the cells were positive for CD29 and CD44 immunofluorescence staining. MTT method revealed the absorbance value of PRP group at 1, 2, 3, 4 and 5 days was 0.137 ± 0.015, 0.219 ± 0.023, 0.367 ± 0.031, 0.586 ± 0.039 and 0.948 ± 0.046, respectively, and in the control group, it was 0.081 ± 0.009, 0.115 ± 0.012, 0.162 ± 0.017, 0.242 ± 0.025 and 0.356 ± 0.032, respectively, suggesting there were significant differences between two groups (P lt; 0.01). At 7 days after osteogenic induction, PRP group was positive for ALP staining, grey-black cell plasm and black precipitate were evident; the positive cells increased
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 study the technique and method of urethral epithelium culture in vitro, so as to lay the groundwork for reconstructing a tissue engineering urethra and to provide an experimental model of urethral mucosa in physiological, pathological, toxicological and microbiological study. METHODS: The urethral mucosa from a young male New Zealand hare that had just been out of milk, was digested into single cell liquid with Dispase II and mixed enzyme, and the fibroblast were removed. After being seeded, the cells were cultured by using L929 cells as trophoderm. The medium was changed regularly and the cells were subcultured when they grew to mix together 80% to 90%. The cultured cells were analyzed with histochemistry, immunohistochemistry dyeing and flow cytometry examination. We observed the ultrastructure of cells with scanning electron microscope and transmission electron microscope. RESULTS: The primary cultured cells fused when they had been cultured for about ten days. They were the same in size like road rocks. The cultured cells were all epithelial cells without fibroblasts and were diploid cells. The cells could be subcultured 11-13 generations, and could survive 50-60 days. CONCLUSION: The urethral epithelium of young New Zealand hare can be cultured in vitro and maintain the ability to proliferate within a certain time. The study result not only sets a role in reconstructing a tissue engineering urethral mucosa, but also provides an experimental model for the research of urethral mucosa in vitro.
Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.