Objective To study degradation of the antigen-extracted meniscus in PBS solution with no enzyme or with different enzymes. Methods Four types of enzymes (collagenase, hyaluronidase, trypsin, papain) were used to enzymolyze the antigen-extracted meniscus and the fresh meniscus for 3, 7, 15 and 30 days (37℃). The antigenextracted meniscus and the fresh meniscus were immersed in PBS solution (37℃) for 30 days. Weight loss measurement, UV spectrophotometry, and scanning electron microscopy (SEM) were used to characterize the degraded materials. Results The two types of the materials were remarkably digested under the enzymes, especially under trypsin. The degradation curves showed that the antigen-extracted meniscus was enzymolyzed less than the fresh meniscus. The degradation products were grouped as amino, peptide, and polyose by the analysis. Both of the materials could hardly behydrolyzed in PBS solution without the enzymes. The four different enzymes had different surface morphologies under the examination of SEM. Conclusion The antigen-extracted meniscus is enzymolyzed more slowly than the fresh meniscus in vitro, and the result can be used as a guideline to the further research.
Objective To compare the myogenic differentiation abil ity in vitro of rabbit adipose-derived stem cells (ADCSs) from different sites so as to provide ideal seed cells for repair and reconstruction of urinary tract. Methods Adipose tissues were obtained from the nape of the neck, post peritoneum, and vicinity of epididymis of a 4-month-old male New Zealand rabbit and ADSCs were harvested through collagenase digestion. ADSCs were purified by differential attachment method. The protein marker CD44 of rabbit ADSCs was used to identify the stem cells by immunocytochemistry, then the5th generation of ADSCs were induced to differentiate into adipogenic, osteogenic, and myogenic cells. Multi- differentiation was confirmed by Oil red O staining, von Kossa staining, and RT-PCR. Myogenic differentiation abil ities of ADSCs from 3 different sites were compared between the control group (L-DMEM medium containing 10%FBS) and the experimental group (myogenic medium) by RT-PCR method. Results ADSCs could be easily isolated from adipose tissues of the nape of the neck, post peritoneum, and vicinity of epididymis. ADSCs displayed a typical cobblestone morphology. Brown particles could be seen in ADSCs by CD44 immunocytochemistry staining. Oil red O staining showed red fat drops in ADSCs after 14 days of adipogenic culture. Black matrix could be seen in ADSCs by von Kossa staining after 28 days of osteogenic culture. RT-PCR detection showed moderate α-actin expression in the control group and b α-actin expression in the experimental group after 42 days of myogenic culture. The growth rate of α-actin from the adipose tissue of post peritoneum (28.622% ± 4.879%) was significantly lower (P lt; 0.05) than those from the adipose tissues of the nape of the neck (35.471% ± 3.434%) and vicinity of epididymis (38.446% ± 4.852%). Conclusion The ADSCs from different sites show different myogenic differentiation abil ities in vitro. ADSCs from the adipose tissues of the nape of the neck and vicinity of epididymis can be used as ideal seed cells for tissue engineering of lower urinary tract.
ObjectiveTo investigate the effect of in vitro fenestration on reconstruction of left subclavian artery in endovascular treatment of aortic dissection.MethodsA total of 89 patients with aortic dissection involving left subclavian artery were treated by endovascular treatment in the Second Affiliated Hospital of Fujian Medical University from February 2017 to January 2020. There were 44 patients in the test group, including 36 males and 8 females, with an average age of 58.02±13.58 years. There were 45 patients in the control group, including 35 males and 10 females, with an average age of 54.10±12.32 years. The left subclavian artery was reconstructed by in vitro fenestration in the test group and by chimney technique in the control group. The clinical data were compared between the two groups.ResultsThe operation time of the test group was longer than that of the control group (126.16±7.53 min vs. 96.49±6.52 min, P<0.01). The median follow-up time was 31 (13-48) months. The incidence of endoleak in the test group (4.7%) was lower than that in the control group (18.6%, P=0.04) during the follow-up. There was no statistical difference in the incidence of stroke, myocardial infarction, false lumen thrombosis, retrograde aortic dissection or left subclavian artery occlusion between the two groups (P>0.05).Conclusion In vitro fenestration for reconstructing left subclavian artery in thoracic endovascular aortic repair of aortic dissection is safe and feasible, which is worthy of further clinical promotion.
Objective To explore the relationship between Beclin-1 and the development of pancreatic ductal adenocarcinoma (PDAC). Methods ① Twenty-five PDAC specimens and 20 matched adjacent normal pancreatic tissues were obtained after radical surgery between April 2009 and November 2009 in West China Hospital of Sichuan University. Beclin-1 mRNA and protein expressions were examined by using real-time PCR and immunohistochemistry, respectively. Correlations between expressions of Beclin-1 protein with clinical data of PDAC patients were evaluated. ② PDAC cells were divided into 2 groups, cells of transfection group were transfected with PLenO-WPI-Beclin-1 vector, and cells of non-transfection group didn’t transfected with PLenO-WPI-Beclin-1 vector. Expressions levels of Beclin-1 mRNA in the 2 groups were detected by real-time PCR at 24 hours and 48 hours after transfection. ③ PDAC cells were divided into 3 groups, cells of transfection group were transfected with PLenO-WPI-Beclin-1 vector, cells of empty vector group transfected with PLenO-WPI, cells of blank control group didn’t accepted any vector. OD value was detected by MTT once a day during 1–7 days after transfection. Results ① Expression levels of Beclin-1 mRNA and its protein were significantly lower in PDAC tissue than those of adjacent normal pancreatic tissues (P<0.05). Increased Beclin-1 expression was associated with early TNM stage of Ⅰ and Ⅱ(P<0.05) and negative distant metastasis (P=0.011). ② At the same time point of 24 hours and 48 hours after transfection, the expression levels of Beclin-1 mRNA were higher in transfection group than those of non-transfection group (P<0.05). ③ MTT assay showed that PANC-1 cell proliferation ability was lower in the transfection group compared to the blank control group and empty vector groups in vitro on day 4–7 after transfection (P<0.05), but there was no significant in the cell proliferation ability among the 3 groups on day 1, 2, and 3 (P>0.05). Conclusions Down regulation of Beclin-1 and autophagy inhibition play an important role in the tumorigenesis and development of PDAC. Activating autophagy via overexpression of Beclin-1 may be a potential treatment for some PDACs and warrants further investigation.
Following the peritendon was removed by means of microsurgical technique, the tenocyte was isolated from the human embryonic tendons by digesting it with trypsin and collagenase. These cells were all stored in frozen condition until they were cultured by F12 culture fluid added with 20% FBS to the 15th generation.These cells were able to grow adhering to the wall and stop growing with contact inhibition. The time of cellsgroup duplication was 4 days, which was similar to the peak time of its mitosis. The number of its chromosome group 2n=46 was 87.5-91.0%. The optimal conditions for tendon cell culture in vitro were investigated, and it was found that after they were reaminated and subcultured the frozen storage didn’t influence their growth, morphology, genetic characteristics. In our research we detected the content generation cells and found the cultured human embryonic tenocyte had same ability never changed with the cells subcultured. We also disscussed the future of tenocyte-a biomaterial in the field of artificial implant.
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.
ObjectiveTo observe the effects of different concentrations of trastuzumab alone or in combination with oxaliplatin on proliferation, apoptosis, and cell cycle of SW-620 human colon cancer cell, and to explore its mechanism. MethodsSW-620 human colon cancer cells were cultured in vitro. ① Cell proliferation experiment: the cells were divided into two large groups: trastuzumab group and trastuzumab combined with oxaliplatin group. There were eight concentration groups in each large group (five holes for each group). The concentration of the trastuzumab group was 0, 0.001, 0.01, 0.1, 1, 10, 100, and 1 000 μg/mL, corresponding to the trastuzumab combined with oxaliplatin group. The concentration of the antibiotic was the same as before, except that oxaliplatin (10 μmol/L) was added. The absorbance (A) value of each group was measured by CCK-8 method. ② Apoptosis experiment: the same proliferation experiment was performed in the group, except that the concentrations of trastuzumab only included 0, 0.1, 1 and 10 μg/mL. Flow cytometry was used to detect the proportion of apoptotic cells and cell cycle distribution in each group. ③ Determination of human epidermalgrowth factor receptor-2 (her-2). The SW-620 cells were divided into two large groups, the concentration of trastuzumab group concluded 0, 100, and 1 000 μg/mL, as well as the concentration of trastuzumab in the trastuzumab combined with oxaliplatin group concluded 0, 1, and 10 μg/mL. Expressions of her-2 protein in SW-620 cells were detected by Western blot method. Results① Cell proliferation assay: the A values at100 μg/mL and 1 000 μg/mL were significantly lower than that at 0 μg/mL (P<0.05). At the same concentration, the A value of the trastuzumab combined with oxaliplatin group was lower than that of the trastuzumab group (P<0.05 ), and the A value gradually decreased with the increase of the concentration of trastuzumab. ② Apoptosis experiment: the proportion of apoptotic cells in the trastuzumab combined with oxaliplatin group was higher than that in the trastuzumab group (P<0.05) at the same concentration of trastuzumab. Flow cytometry: after treatment with different concentrations of trastuzumab combined with oxaliplatin, cells in G1 phase showed a downward trend, and cells in S phase showed an upward trend in a dose-dependent manner. At 1 and 10 μg/mL concentration of trastuzumab, the trastuzumab combined with oxaliplatin group significantly reduced the proportion of cells in the G1 phase of SW-620 cell cycle compared with the trastuzumab group, but S phase ratio was higher (P<0.05). The proportion of G2 phase cells was significantly higher in the trastuzumab combined with oxaliplatin group than the trastuzumab group at 0.1, 1 and 10 μg/mL concentrations of trastuzumab (P<0.01). ③ Expressions of her-2 protein: the expression level of her-2 protein gradually decreased at 1, 100, and 1 000 μg/mL trastuzumab group (P<0.05). The expression levels of her-2 protein in 0, 1 and 10 μg/mL trastuzumab combined with oxaliplatin group also gradually decreased (P<0.01). ConclusionsHigh concentration of trastuzumab can inhibit the proliferation of SW-620 human colon cancer cells and induce apoptosis. Trastuzumab combined with oxaliplatin has synergistic effect on inhibiting cell proliferation and promoting apoptosis.
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 investigate the way and process of degradation behavior of acellular porcine aortic valve in vitro. Methods Acellular porcine aortic valve（n=90）were randomly divided into 3 groups （collagenase group, elastase group, control group）, 30 piece in each group . Behavior of acellular porcine aortic valve was degradated with 0.05mg/ml collagenase Ⅰ, 0. 05mg/ml elastase, phosphate buffered solution in collagenase group, elastase group and control group. The histomorphology, weight loss, value of protein and hydroxyproline were observed at 3,6,9, 12, 15 and 30d after degradation. Results The behavior of acellular porcine aortic valve of collagenase group and elastase group became poorer, looser and broken gradually in degradation. The weight loss of valve, the value of protein and hydroxyproline in vehiculum became greater gradually in collagenase group and elastase group（P〈0. 01）. Furthermore the effect of collagenase Ⅰ was b than elastase in degradation. Conclusion The effect of collagenase Ⅰ and elastase can degradate the acellular porcine aortic valve in vitro. Collagenase Ⅰ is b than elastase in degradation.
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.