目的：机械分离、培养小鼠耳蜗螺旋神经元，并进行免疫荧光细胞学鉴定，为后期进一步的实验研究提供实验材料。方法：采用初出生1～5天以内的昆明小鼠进行解剖、机械分离以获得螺旋神经节组织，进行原代培养后，应用神经微丝蛋白（Neurofilament protein,NFP－H）单克隆抗体进行免疫荧光细胞学鉴定。结果：机械分离后获得的螺旋神经节组织中的螺旋神经元，在体外培养条件下可以存活并进行正常分化。典型的螺旋神经元，其细胞形态呈椭圆形,胞体透明光滑、接近生理形态。荧光染色标记后，胞体和神经突起均显色好，Schwann细胞和成纤维细胞未着色。结论：应用机械分离的方法获得小鼠耳蜗螺旋神经节组织并进行培养，耳蜗螺旋神经元在体外可以稳定地存活生长。培养获得的细胞形态和生存状态接近生理状态，满足电生理、免疫细胞化学、药理学等研究。应用特异性的神经微丝蛋白对培养获得的螺旋神经元进行免疫荧光细胞学鉴定，特异性好，荧光显色好。
The effects of pentagastrin (PG) on the viable cell count (Α value) and the synthesis of DNA (CPM value) of primary cultured large bowel carcinoma cells in 25 patients were evaluated in vitro by MTT assay,3H-TdR incorporation. The results showed that Α value and CPM value in well， moderately and poorly-differentiated carcinoma cells were higher than normal control (Ｐlt;0.01，Ｐ＜0.05). The proliferative effect was significant at a dose of 0.3907 μg/ml in well-differentiated carcinoma cells, and at a dose of 6.2500μg/ml in moderately and poorly-differentiated carcinoma cells. These indicat that PG has the proliferative effect on large bowel carcinoma cells. These results provide an experimental foundation for the endocrine therapy for patients with large intestine carcinoma, especially by using gastrin receptor antagonists for well-differentiated carcinoma.
Objective To predict clinical chemotherapy sensitivity of primary non-small cell lung cancer（NSCLC） by methylthiazal （MTT） tumor chemosensitivity assay method in vitro and detection of multidrug resistance gene1 （MDR1）, and provide reference for clinical individualized treatment. Methods We selected 80 fresh primary NSCLC samples from NSCLC patients who underwent surgical resection in Zibo Central Hospital Affiliated to Binzhou Medical College between January 2009 and December 2011. There were 46 male patients and 34 female patients with their median age of 54 （29 to 81）years. Viable NSCLC cells obtained from malignant tissue were tested for their sensitivity to cisplatin （DDP）, gemcitabine （GEM）, docetaxe （DOC）, etoposide （VP-16） ,and vinorelbine （NVB） using MTT assay in vitro. Fluorescent quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） was used to analysis the expression level of multidrug resistance gene1 （MDR1）. Results After exposure to antitumor drugs, morphologic changes, decrease of metabolic activity, and apoptosis were detected in NSCLC cells. MTT results showed that different individual cancer cells had different chemosensitivity to antitumor drugs, and cancer cells also had different chemosensitivity to different antitumor drugs. Inhibitory rates of cancer cells exposed to DOC, GEM, and VP-16 were significantly higher than those of cancer cells exposed to DDP and NVB （42.5%±9.5%, 40.5%±6.5%, 38.4%±7.6% versus 31.5%±8.5%，32.5%±7.8%, P＜0.05）.The positive rate of MDR1 in tumor tissues was 40.0% （32/80）. The expression of MDR1 was not associated with tumor histological type, degree of differentiation, lymph node metastasis and TNM stage. The expression of MDR1 was associated with resistance to NVB （χ2=5.209，P=0.022），GEM （χ2=4.769，P=0.029），VP-16 （χ2=4.596，P=0.032），and DDP（χ2=6.086，P=0.014）, but not associated with resistance to DOC（χ2=0.430，P=0.512）. Conclusion MTT chemosensitivity assay can effectively predict clinical chemotherapy sensitivity. Detection of MDR1, together with MTT chemosensitivity assay, can more accurately predict NSCLC chemosensitivity and be a guide for individualized chemotherapy of NSCLC.
目的：通过体外研究探讨化疗剂量对乳腺癌治疗效果的影响。方法：通过手术取材和肿瘤组织原代细胞体外培养技术，对96例乳腺癌患者进行体外药物敏感性检测。结果:乳腺癌晚期患者比早期患者的耐药风险明显增加（P＜0.05），乳腺癌复发患者比原发患者具有更强的多药耐药能力（P＜0.01）；增加化疗药物剂量仍然可以提高大多数耐药患者的治疗效果（P＜0.01）。结论：大剂量化疗方案对晚期和复发性乳腺癌患者有帮助。
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.
This study was aimed to explore the methodology regarding culture, proliferation and purification of adipose-derived stromal cells (ADSCs), and to study their biological characteristics. ADSCs were obtained using type Ⅰ collagenase digestion method. Cell growth was observed, and cell viability were detected under different digestion period by MTT. The ADSCs were then identified and induced. The results showed that adherent cells digested by type Ⅰ collagenase for 60 min had a strong proliferation capability. After the induction of different inducers these adherent cells could differentiate into nerve cells and fat cells. The best digestion period was proved to be of 60 minutes in the experiment. The results indicate that stem cells with multilineage differentiation ability could be separated from adipose tissue, namely ADSCs.
ObjectiveTo explore a simple, efficient method for primary culture in vascular smooth muscle cells of diabetic rat, to establish a long-term stable diabetic vascular smooth muscle cell model in vitro, and lay the foundation for the study of diabetes chronic vascular lesions. MethodsTwenty diabetic Wister rats models were self-made by using streptozotocin intraperitoneal injection plus high fat and sugar feeding, the vascular smooth muscle cells in vitro was cultured by a modified enzymatic digestion. ResultsThe diabetic rat models were successfully established, the vascular smooth muscle cells cultured in vitro by modified enzyme digestion grew fast, the cell survival was 96%, it could meet the requirements for cell experiment in vitro. ConclusionComparing with the traditional method, the modified enzymatic digestion method is simple, economic, high survival rate.
ObjectiveTo establish a method of air-liquid interface culture and ciliary beat frequency measurement of mouse tracheal-bronchial epithelial cells to simulate the physiological function of airway epithelium.MethodsBALB/c mouse tracheal-bronchial epithelial cells were obtained by digestion with 1 mg/mL protease in cold temperature overnight, and the digestion time was optimized to ensure the quantity and viability of the obtained cells. After removing fibroblasts by differential velocity adhesion method, the cells were cultured into collagen coated Transwell inserts. Proliferating phase and air-liquid interface culture were promoted with different culture media.ResultsCell numbers obtained by cold protease overnight digestion for 12 h, 14 h and 16 h were (1.78±0.33)×105, (1.93±0.26)×105 and (2.01±0.28)×105, respectively. Cell viability by trypan blue staining were (96.86±0.25)%, (94.73±1.63)% and (86.87±5.95)%, respectively. Cells were 100% confluent in Transwell chamber after 1-week proliferation, and the ciliary beat frequency was observed under microscope after 2 - 3 weeks of air-liquid interface culture. The cilia structure was confirmed by hematoxylin-eosin staining, electron microscopy and immunofluorescence. Ciliary beat frequency of the cells obtained by this method was consistent with that of mouse trachea in vivo, which further demonstrated its capacity in simulating the physiological function of airway epithelium. ConclusionsThe separation and air-liquid interface culture system as well as the ciliary beat frequency measurement method established in this experiment is simple, stable, efficient and reliable, which establishes a substantial foundation for exploring the pathogenesis and treatment mechanism of airway diseases. It can also provide reference for the culture of epithelium in the airway of other species and/or other organs.
ObjectiveTo optimize the culture method of human primary pancreatic ductal adenocarcinoma (PDAC) cells and cancer associated fibroblasts (CAFs) and investigate the effect of CAFs on the growth of primary PDAC cells in vitro and tumor formation in patient-derived xenograft (PDX) model.MethodsThe PDAC specimens were collected and primarily cultured. In order to observe the effect of CAFs on the growth of primary PDAC cells in vitro, the CAFs were co-cultured with primary PDAC cells consistently and the alone cultured primary PDAC cells served as the control. Then, these cells were injected into the shoulder blades of NOG mice in order to develop the PDX model.ResultsWhen the primary PDAC cells separated from the CAFs, the proliferation capacity of the primary PDAC decreased rapidly in the passage culture in vitro, and the most cells were terminated within 5 generations. By contrast, when the CAFs co-cultured with the primary PDAC cells, the proliferation capacity of primary PDAC cells were preserved, which could be stably transferred to at least 10 generations. The tumors of NOG mice were detected during 2–3 weeks after injecting the mixed cells (primary PDAC plus CAFs), while had no tumor formation after injecting CAFs alone. The rate of tumor was 92.9% (13 cases) in the primary PDAC plus CAFs group, which was higher than that of the CAFs alone group (64.3%, 9 cases), but there was no statistical difference because of the small sample size. The volume of tumor in the primary PDAC plus CAFs group at 2, 4, 6, and 8 weeks after the tumor cells injection was significantly larger than that in the CAFs alone group at the corresponding time point, the differences were statistically significant (P<0.01).ConclusionsThe CAFs could promote the growth of primary PDAC cells in vitro. This new method of co-culture CAFs with primary PDAC could improve the success rate of primary PDAC cells culture and improve the success rate of PDX model in NOG mice.