Design

An in vitro, comparative, observational study of cytology.

Time and setting

The experiment was performed at the Laboratory of Human Anatomy and Histology and Embryology, Weifang Medical University, China from 2009 to 2011.

Materials

Twelve 6-week-old Sprague-Dawley rats, of either gender, weighing 150–180 g, of clean grade, were provided by the Animal Research Center, Weifang Medical University, license No. SYXK (Lu) 2005 0043. Animals were housed with illumination periods from 7:00–19:00, at 20 ± 2°C, with a relative humidity of 45–55%, and allowed free access to food and water. Rats were handled in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[36].

Methods

Culture of rat bone marrow-derived mesenchymal stem cells

Rat bone marrow-derived mesenchymal stem cells were cultured using their physical characteristic of adhering to a plastic surface^[37]. Rats were sacrificed by cervical dislocation, following which tibias and femurs were obtained under sterile conditions. After cutting away the ends of the bones, the marrow (5 mL) was extracted using a needle and syringe. Cells were suspended in Dulbecco’s-modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) at 37°C in a humidified- atmosphere of 95% air and 5% CO₂. After 24 hours, nonadherent cells were removed by replacing the medium. The medium was changed every 3 days. Once more than 80% of adherent cells converged, they were detached with trypsin (Sigma, St. Louis, MO, USA). To expand the culture, the cells were diluted at 1:2 per passage. Passage 3 cells were used for neuronal induction and subsequent determination of their biological identity.

Dopaminergic neuronal induction of bone marrow-derived mesenchymal stem cells

Passage 3 bone marrow-derived mesenchymal stem cells were trypsinized and subcultured onto polylysine-coated coverslips in multiwell plates. Each plate contained 1 × 10⁵ cells. At approximately 80% confluence, bone marrow-derived mesenchymal stem cells were induced into dopaminergic neurons using different inducers as follows: in the Xiangdan injection group, cells were pre-induced in L-Dulbecco’s-modified Eagle’s medium containing 10% fetal bovine serum and 25 ng/mL basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, USA) for 24 hours, followed by L- Dulbecco’s-modified Eagle’s medium and 20% Xiangdan injection (containing 1 000 g/L salvia miltiorrhiza,       1 000 g/L dalbergia odorifera (No. Z13021387), Hebei Tiancheng Pharmaceutical Co., Ltd., China) for 3 hours; in the all-trans retinoic acid + glial cell line-derived neurotrophic factor group, cells were induced in neurobasal medium containing 25 ng/mL basic fibroblast growth factor and 2% B27 (Gibco) for 24 hours, followed by neurobasal medium containing 2% B27, 1 μM all-trans retinoic acid (Sigma) and 50 ng/mL glial cell line-derived neurotrophic factor (Peprotech) for 6 days; in the sonic hedgehog + fibroblast growth factor 8 group, cells were induced in neurobasal medium containing 25 ng/mL basic fibroblast growth factor and 2% B27 for 24 hours, followed by neurobasal medium containing 2% B27,  250 ng/mL sonic hedgehog (Peprotech) and 100 ng/mL fibroblast growth factor 8 (Peprotech) for 12 days; in the control group, cells were cultured without any inducers.

Immunocytochemistry and immunofluorescence for detection of neural proteins

Cells were fixed with acetone at 4°C for 10 minutes, incubated in 3% H₂O₂-methanol for 30 minutes and washed three times with 0.01 M PBS. For blocking nonspecific immune reaction, the cells were treated with goat serum at room temperature for 30 minutes. Cells were incubated overnight at 4°C with the following antibodies: rabbit anti-CD34 polyclonal antibody (1:300; Wuhan Boster, Wuhan, China); rabbit anti-CD44 polyclonal antibody (1:300; Wuhan Boster); rabbit anti-nestin polyclonal antibody (1:100; Wuhan Boster); mouse anti-rat microtubule-associated protein 2 monoclonal antibody (1:100; Wuhan Boster); rabbit anti-neuron specific enolase polyclonal antibody (1:100; Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd. Beijing, China); rabbit anti-glial fibrillary acid protein polyclonal antibody (1:100; Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd.); mouse anti-rat tyrosine hydroxylase monoclonal antibody (1:500; Sigma); goat anti-vesicular monoamine transporter-2 polyclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three washes in 0.01 M PBS, cells were incubated with biotinylated goat anti-mouse or anti-rabbit IgG (General SP Kit working fluid, Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd.) at 37°C for 30 minutes, followed by 30 minutes of incubation in avidin-biotinylated peroxidase complex at 37°C, except for the vesicular monoamine transporter-2. For vesicular monoamine transporter-2, the cells were treated with the polymer auxiliary agent at 37°C for 30 minutes, and then mixed with horseradish enzyme labeled rabbit anti-goat IgG dimmer at 37°C for 30 minutes. After washing with 0.01 M PBS, diaminobenzidine (Zhongshan Golden Bridge Biological Technology Co., Ltd.) was used as a chromagen, with reactions sustained at room temperature and in the dark for 10 minutes. After decoloration with distilled water, cells were counterstained with hematoxylin, dehydrated with a gradient alcohol series, cleared with dimethyl benzene and mounted with neutral balsam. The primary antibodies in the negative control group were replaced by PBS. The expression of each antigen was examined in separate experiments at least three times. Ten nonrepetitive visual fields were selected randomly under a light microscope in each group (× 100). Expression rate (%) = the number of positive cells/(the number of positive cells + the number of negative cells) × 100%.

For immunofluorescence, cells were incubated overnight at 4°C with the following antibodies: mouse anti-rat tyrosine hydroxylase monoclonal antibody (1:250) regularly mixed with rabbit anti-neuron-specific enolase polyclonal antibody (1:50). After rinsing with 0.01 M PBS for three times, cells were incubated with secondary antibodies: fluorescein isothiocyanate-goat anti-mouse IgG (1:100, Zhongshan Golden Bridge Biological Technology Co., Ltd.) regularly mixed with Cy3-goat anti-rabbit IgG (1:500, Beyotime, Shanghai, China) at 37°C for 60 minutes in the dark. Cell nuclei were counterstained with Hoechst 33258 (Beyotime) at room temperature for 10 minutes in the dark. Labeled cells were observed under a fluorescent microscope (Leica, Solms, Germany) with the appropriate fluorescence filters.

Measurement of catecholamine levels in supernatant fluid

After induction, the supernatant fluid was collected, centrifuged at 2 000 r/min and 0.25 g EDTA-Na₂ was then added, shaken, and boiled for 2 minutes. After cooling, they were placed in 0.75 g Al₂O₃, shaken for 2 minutes, mixed with 10 M NaOH until the pH reached 8.0–8.6, and shaken for 5 minutes. The pH was re-adjusted between 8.0–8.5, and the product was shaken for 5 minutes again. Al₂O₃ was transferred to the column, washed with sodium acetate and double distilled water, followed by 2 mL of  0.5 M H₂SO₄. The eluent in burettes was gathered after elution twice and then formulated for testing. The catecholamine level was detected using the fluorescence spectrophotometer RF-5301PC (Shimadzu, Kyoto, Japan), with excitation and emission wavelengths of 410 nm and 510 nm, respectively.

Statistical analysis

The SPSS 16.0 software (SPSS, Chicago, IL, USA) was used to analyze data, which were expressed as mean ± SEM. Statistical differences were calculated using F-test. P values < 0.05 were considered statistically significant.

1 This study compared the efficiency of the induction of bone marrow-derived mesenchymal stem cells to differentiate into dopaminergic neurons using injection of the traditional Chinese medicine, Xiangdan, and a combination of inducing agents (all-trans retinoic acid + glial-derived neurotrophic factor) and cytokines (sonic hedgehog + fibroblast growth factor 8).
2 Based on the expression of tyrosine hydroxylase and the levels of catecholamines in the induced cells, all three combinations examined could induce dopaminergic neuronal differentiation from bone marrow-derived mesenchymal stem cells, including the Xiangdan injection.
3 The combination of sonic hedgehog + fibroblast growth factor 8 had the highest induction efficiency.
1 比较了中药诱导剂丹参、诱导分化剂组合（全反式维甲酸+胶质细胞源性神经营养因子）和细胞因子组合(音速波状蛋白+成纤维细胞生长因子8)对骨髓间充质干细胞向多巴胺能神经元的诱导效率。
2 基于诱导后细胞酪氨酸羟化酶表达和儿茶酚胺含量，认为碱性成纤维细胞生长因子+香丹注射液、碱性成纤维细胞生长因子+全反式维甲酸+胶质细胞源性神经营养因子、碱性成纤维细胞生长因子+音速波状蛋白+成纤维细胞生长因子8均能将骨髓间充质干细胞诱导分化为多巴胺能神经元。
3 碱性成纤维细胞生长因子+音速波状蛋白+成纤维细胞生长因子8组具有较高诱导效率。

1 中文内容介绍
通过大鼠骨髓间充质干细胞体外培养、传代，将第3代骨髓间充质干细胞经碱性成纤维细胞生长因子预诱导24h后，分别采用3种方法诱导其向多巴胺能神经元分化。A组：香丹注射液；B组：全反式维甲酸+胶质细胞源性神经营养因子；C组：音速波状蛋白+成纤维细胞生长因子8。采用免疫细胞化学和免疫荧光方法检测诱导后细胞神经系统蛋白如巢蛋白、神经元特异性烯醇化酶、微管相关蛋白2、胶质纤维酸性蛋白、酪氨酸羟化酶、囊泡单胺转运体的表达，检测细胞周期相关蛋白增殖细胞核抗原、Bcl-2、细胞周期蛋白依赖性激酶、CyclinB1的表达变化。检测各组诱导细胞上清液中儿茶酚胺含量，以确定诱导后细胞的分泌功能。结果：各组细胞诱导后均呈典型的神经元样形态，C组细胞诱导后呈高水平表达巢蛋白, 神经元特异性烯醇化酶, 微管相关蛋白2, 酪氨酸羟化酶 和 囊泡单胺转运体，且酪氨酸羟化酶诱导率高达46.7±3.3%，说明C组诱导效率高。与对照组比较，3个诱导组细胞表达增殖细胞核抗原, 细胞周期蛋白依赖性激酶和 Cyclin B1显著下调。3个诱导组上清中儿茶酚胺含量分别为2.487±0.43μg/L，2.731±0.81μg/L和2.977±0.16 μg/L，进一步表明C组的诱导效率显著高于其他2组。