Induced pluripotent stem cell-related approaches to generate dopaminergic neurons for Parkinson's disease

The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease,the second most common human neurodegenerative disease.Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear,the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy.The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons,which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies.The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells.The benefits of induced pluripotent stem cell-based research are highlighted.Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared.The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated.Finally,limitations,challenges,and future directions of induced pluripotent stem cell–based approaches are analyzed and proposed,which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.

Bis(7)-Tacrine, a Promising Anti-Alzheimer's Agent,Attenuates Glutamate-Induced Cell Injury in Primary Cultured Cerebrocortical Neurons of Rats

The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7) tacrine (0.03 1.0 μmol/L) reduced the glutamate induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection. A receptor binding assay showed that bis(7) tacrine can completely displace MK 801 binding to rat cortical membrane with an IC 50 of 0.57 μmol/L. These findings suggest that bis(7) tacrine can directly interact with N methyl D aspartate receptor channel complex, which may contribute to the inhibitor's protective effects against glutamate induced excitotoxicity. Thus, it is possible that anti glutamate/anti AChE synergism is responsible for potentially better Alzheimer's therapy of bis(7) tacrine relative to tacrine.

MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.

3D Collagen Gels:A Promising Platform for Dendritic Cell Culture in Biomaterials Research

The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.

Dose-dependent effects of lead on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons

BACKGROUND： Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain poorly understood. OBJECTIVE： To investigate the effects of different lead concentrations on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons. DESIGN, TIME AND SETTING： A randomized, controlled, in vitro experiment was performed at the China Medical University between July 2008 and May 2009. MATERIALS： Antibodies specific to cyclin D1 and actin were synthesized and purified by Santa Cruz Biotechnology, USA. FACStar flow cytometer was purchased from Becton Dickinson, San Jose, California, USA. METHODS： Wistar rat hippocampal neurons were primary cultured for 7 days. Neurons in the control group were treated with 0.01 mol/L phosphate buffered saline. Neurons in the 0.2, 1.0, and 10 umol/L lead acetate groups were subjected to 0.2, 1.0, and 10 umol/L lead acetate. Subsequently hippocampal neurons in each group were cultured for 24 hours. MAIN OUTCOME MEASURES： The effects of lead on cell cycle were measured by flow cytometry, DNA damage was measured using the comet assay, and cyclin D1 expression was measured using Western blot analysis. RESULTS： Treatment of hippocampal neurons with 0.2 umol/L lead acetate did not significantly alter cell cycle phase distribution, i.e., sub-G1, S, G0/G1, G2/M, whereas treatment with 1.0 and 10 umol/L lead acetate significantly increased the percentage of S and sub-G1 phase cells （P 〈 0.05）. Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 umol/L and 10 umol/L lead acetate groups were significantly greater compared with the control group （P 〈 0.05）. In addition, the percentage of tail DNA was greater in the 0.2 umol/L lead acetate group compared with the control group （P 〉 0.05）. Following incubation with 0.2, 1.0, and 10 umol/L lead acetate for 24 hours, cyclin D1 expression gradually decreased with exposure to increasing lead acetate concentrations （1.0-10 umol/L）. CONCLUSION： Lead exposure to primary cultured rat hippocampal neurons resulted in dose-dependently disturbed cellular homeostasis, including DNA damage, reduced cyclin D1 expression, and stagnation of cell-cycle progression.

Chemical Constituents of the Suspension Cell Cultures of Maytenus hookeri

Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.

Detection of Mycoplasma Contamination in Animal Cell Cultures

[ Objective ] To develop a rapid efficient method for detecting mycoplasma contamination in cell cultures. [ Method] A pair of primers was designed according to two highly conserved nucleotide sequences of the 16S RNA from six kinds of mycoplasma that commonly contaminated cells. Then the mycoplasma contamination of 25 cell samples was defected by PCR and DNA fluorescence staining. EResultl When these cell samples were detected by DNA fluorescence staining, the positive rate and probable positive rate were respectively 24% and 16%. And when they were detected by PCR, the positive rate was 36%. [ Condusion] The PCR method is more sensitive and specific than the DNA fluorescence staining, and combining these two methods is the optimal way to detect mycoplasma contamination in cell cultures.

Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro.However,because of the short survival time of the differentiated cells,clinical applications for this technique are limited.As such,we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy.The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended.Taken together,these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death.However,the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

Adult adipose-derived stromal cells differentiate into neurons with normal electrophysiological functions

β-mercaptoethanol was used to induce in vitro neuronal differentiation of adipose-derived stromal cells. Within an 8-hour period post-differentiation, the induced cells exhibited typical neuronal morphology, and expression of microtubule-associated protein 2 and neuron-specific enolase, which are markers of mature neurons, reached a peak at 5 hours. Specific organelle Nissl bodies of neurons were observed under transmission electron microscopy. Results of membrane potential showed that fluorescence intensity of cells was greater after 5 hours than adipose-derived stromal cells prior to induction. In addition, following stimulation with high-concentration potassium solution, fluorescence intensity increased. These experimental findings suggested that neurons differentiated from adipose-derived stromal cells and expressed mature K^＋ channels. In addition, following stimulation with high potassium solution, the membrane potential depolarized and fired an action potential, confirming that the induced cells possessed electrophysiological functions.

Differentiation of embryonic versus adult rat neural stem cells into dopaminergic neurons in vitro

BACKGROUND：It has been reported that the conversion of neural stem cells into dopaminergic neurons in vitro can be increased through specific cytokine combinations. Such neural stem cell-derived dopaminergic neurons could be used for the treatment of Parkinson’s disease. However, little is known about the differences in dopaminergic differentiation between neural stem cells derived from adult and embryonic rats. OBJECTIVE： To study the ability of rat adult and embryonic-derived neural stem cells to differentiate into dopaminergic neurons in vitro. DESIGN： Randomized grouping design. SETTING： Department of Neurosurgery in the First Affiliated Hospital of Sun Yat-sen University. MATERIALS： This experiment was performed at the Surgical Laboratory in the First Affiliated Hospital of Sun Yat-sen University （Guangzhou, Guangdong, China） from June to December 2007. Eight, adult, male, Sprague Dawley rats and eight, pregnant, Sprague Dawley rats （embryonic day 14 or 15） were provided by the Experimental Animal Center of Sun Yat-sen University. METHODS： Neural stem cells derived from adult and embryonic rats were respectively cultivated in serum-free culture medium containing epidermal growth factor and basic fibroblast growth factor. After passaging, neural stem cells were differentiated in medium containing interleukin-1α, interleukin-11, human leukemia inhibition factor, and glial cell line-derived neurotrophic factor. Six days later, cells were analyzed by immunocytochemistry and flow cytometry. MAIN OUTCOME MEASURES： Alterations in cellular morphology after differentiation of neural stem cells derived from adult and embryonic rats; and percentage of tyrosine hydroxylase-positive neurons in the differentiated cells. RESULTS： Neural stem cells derived from adult and embryonic rats were cultivated in differentiation medium. Six days later, differentiated cells were immunoreactive for tyrosine hydroxylase. The percentage of tyrosine hydroxylase positive neurons was （5.6 ± 2.8）% and （17.8 ± 4.2）% for adult and embryonic cells, respectively, with a significant difference between the groups （P 〈 0.01）. CONCLUSION： Neural stem cells from embryonic rats have a higher capacity to differentiate into dopaminergic neurons than neural stem cells derived from adult rats.

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals. OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease. DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007. MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA. METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium. MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESULTS： Cell counts of tyrosine hydroxylase-positive neurons in the dopaminergic neuron ＋ PNS group were （732±82.6） cells/400-fold field. This was significantly greater than the dopaminergic neuron group [（326 ± 34.8） cells/400-fold field, P 〈 0.01]. Compared to the control group, the rotational asymmetry of rats that received dopaminergic neuron transplants was significantly decreased, beginning at 20 days after operation （P 〈 0.01）. Rotational asymmetry was further reduced between 10-60 days post-surgery in the dopaminergic neuron ＋ PNS group, compared to the dopaminergic neuron group （P 〈 0.01）. CONCLUSION： Panax notoginseng saponins can increase survival and effectiveness of dopaminergic neurons differentiated from neural stem cells for transplantation in a rat model of Parkinson＇s disease.

Umbilical cord:an unlimited source of cells differentiable towards dopaminergic neurons

Cell replacement therapy utilizing mesenchymal stem cells as its main resource holds great promise for ultimate treatment of human neurological disorders.Parkinson＇s disease（PD）is a common,chronic neurodegenerative disorder hallmarked by localized degeneration of a specific set of dopaminergic neurons within a midbrain sub-region.The specific cell type and confined location of degenerating neurons make cell replacement therapy ideal for PD treatment since it mainly requires replenishment of lost dopaminergic neurons with fresh and functional ones.Endogenous as well as exogenous cell sources have been identified as candidate targets for cell replacement therapy in PD.In this review,umbilical cord mesenchymal stem cells（UCMSCs）are discussed as they provide an inexpensive unlimited reservoir differentiable towards functional dopaminergic neurons that potentially lead to long-lasting behavioral recovery in PD patients.We also present mi RNAs-mediated neuronal differentiation of UCMSCs.The UCMSCs bear a number of outstanding characteristics including their non-tumorigenic,low-immunogenic properties that make them ideal for cell replacement therapy purposes.Nevertheless,more investigations as well as controlled clinical trials are required to thoroughly confirm the efficacy of UCMSCs for therapeutic medical-grade applications in PD.

In Vitro Invasive Pattern of Hepatocellular Carcinoma Cell Line HCCLM9 Based on Three-dimensional Cell Culture and Quantum Dots Molecular Imaging

Summary： This study aimed to establish a new in vitro three-dimensional （3D） cell culture and use quantum dots （QDs） molecular imaging to examine the invasive behaviors of hepatocellular carcinoma （HCC） cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Wnt3a expression during the differentiation of adipose-derived stem cells into cholinergic neurons

The present study analyzed changes in Wnt3a expression during differentiation of adipose-derived stern cells into cholinergic neurons. Immunocytochemistry and immunofluorescence revealed significantly increased nestin, neuron-specific enolase, microtubule-associated protein 2, and choline acetyltransferase expression in adipose-derived stem cells isolated from Sprague-Dawley rats and cultured in vitro in neural-induced medium. These expressions increased with prolonged induction time. Real-time reverse transcription-PCR and western blot assay results demonstrated significantly increased choline acetyltransferase and Wnt3a protein and mRNA expressions, respectively, in adipose-derived stem cells following induction. Choline acetyltransferase expression positively correlated with Wnt3a protein and mRNA expressions. These results demonstrated that neural-induced medium induced differentiation of adipose-derived stem cells into cholinergic neuronal-like cells, with subsequent increased Wnt3a expression.

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

Correlation of PDCD5 and Apoptosis in Hair Cells and Spiral Ganglion Neurons of Different Age of C57BL/6J Mice

This study examined the expression pattern of programmed cell death 5 (PDCD5) in co-chlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice.Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3,6,9 or 12 months).PDCD5 expression was detected by using immunohistochemistry,real-time PCR and Western blot.Morphological change of the cochleae was also evaluated by using immunoassay.The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice,as well as gradually increased apoptosis of cochlear hair cells and SGNs.In addition,we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing.It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs,and thereby plays a role in the pathogenesis of presbycusis.Thus,PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

Effects of brain-derived neurotrophic factor on synapsin expression in rat spinal cord anterior horn neurons cultured in vitro

Brain-derived neurotrophic factor （BDNF） promotes synaptic formation and functional maturation by upregulating synapsin expression in cortical and hippocampal neurons. However, it remains controversial whether BDNF affects synapsin expression in spinal cord anterior horn neurons. Wistar rat spinal cord anterior horn neurons were cultured in serum-supplemented medium containing BDNF, BDNF antibody, and Hank＇s solution for 3 days, and then synapsin I and synaptophysin protein and mRNA expression was detected. Under serum-supplemented conditions the number of surviving neurons in the spinal cord anterior horn was similar among BDNF, anti-BDNF, and control groups （P 〉 0.05）. Synapsin I and synaptophysin protein and mRNA expressions were increased in BDNF-treated neurons, but decreased in BDNF antibody-treated neurons （P 〈 0.01）. These results indicated that BDNF significantly promotes synapsin I and synaptophysin expression in in vitro-cultured rat spinal cord anterior horn neurons.

Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease

BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.

Evaluation of diffusion in gel entrapment cell culture within hollow fibers

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.