| Background:Parkinson disease (PD) is a degenerative disorder, which is characterized by a loss of midbrain dopamine (DA) neurons with a subsequent reduction in striatal DA. Dopamine replacement is the main treatment method at present. However, this replacement treatment works initially. With the progression of PD, efficacy will be reduced and motor complications developed. Therefore, alternative treatment is required. Stem cells, which have the capacity to self-renew and differentiate into other cells, may provide a solution. Technical and ethical difficulties in obtaining DA neurons derived from embryonic stem (ES) cells, induced pluripotent stem cells, and bone marrow-derived mesenchymal stem cells (MSC) have limited the application of these kinds of stem cells, peripheral blood insulin-producing cells is a novel type of stem cells from human peripheral blood, which can avoid this limitations. To examine in vivo function of peripheral blood insulin-producing cells(PB-IPC),We transplant PB-IPC in a rat model of Parkinson’s disease.Method:1. cell preparationsHuman peripheral blood samples were collected from healthy donors at Jinan Central Hospital. Mononuclear cells were isolated with Ficoll-Hypapue, then red blood cells were removed using red blood cell lysis buffer. The remaining Cells were cultured in serum-free culture medium.2. Quantitative real-time PCRQuantitative real-time PCR was used to quantify mRNA expression for genes related embryonic stem cell. Total RNA was extracted using a Qiagen kit. First-strand cDNAs were synthesized using a High-Capacity cDNA reverse transcription kit according to the manufacturer’s instructions. Real-time PCR was performed on each sample in triplicate with the ABI7300Real-Time PCR System using the follow protocol:50℃,2minutes;95℃,2minutes;65℃,1minute. Expression levels were determined relative to (β-ctin as an internal control. The validated PCR Primer sets for each gene were designed and purchased from Takara Biotechnology.3. Establish a rat model of PDEstablish a rat model of PD by direct injection of6-OHDA in MFB and SNC. To check if the establishment of PD rat model was successive by hypodermical injection of apomorphine.4. Rotational BehaviorThree weeks after receiving the6-OHDA unilateral lesion, the animals were tested for apomorphine-induced rotation (0.5mg/kg, ip). The Animals who exhibited rotations to left and>7rotations/min in a30min period were successful models of Parkinson’s disease. After the transplantation, behavior analysis were conducted at various time points(weeks2,4,6and8).5. ImmunocytochemistryAt8weeks, Rats were deeply anesthetized with5%chloral hydrate anesthesia and transcardially perfused with4%paraformaldehyde. The SNC of Brains were sectioned at20μm thickness on a Leica CM3050S at-20℃.Sections were blocked (10%goat serum,0.5%triton) and incubated with the primary antibody overnight at4℃. Anti-bromodeoxyuridine (brdU) was mixed with other primary antibodies. Then the primary antibodies were incubated with secondary antibodies.6. ELISA testRats were sacrificed at8weeks after transplantation. Brain tissues were removed and with ice-cold PBS (0.02mol/L, pH7.0-7.2) rinse thoroughly the tested tissues, weighing the tissues, and the tissues were made into10%tissue bomogenatel:9proportions (1g tissue blocks:9ml PBS), at4℃5000r/min, centrifuged15min. The supernatants measured for neurotransmitter dopamine levels by ELISA. Results:1. PB-IPC displayed morphological and growing characteristics similar with CB-SCsWe observed that PB-IPC displayed morphological and growing characteristics similar with CB-SCs (Cord blood-derived multipotent stem cells):round or oval and adhering to the bottom of Petri dish tightly2. PB-IPC make an expression of embryonic stem cell related transcription factorsIt has been established that PB-IPC make an apparent expression of Sox2, Nanog, c-Myc, Klf4, Ngnl, Ngn2and Mashl.3. Functional Recovery of apomorphine-Induced Motor Asymmetry in a Rat Model of PDThe rotation scores of PD rats in the Transplantation group by the apomorphine irritation were reduced consistently after transplantation (26±2%at the2th week,34±4%at the4th week,39±7%at the6th week,37±5%at the8th week); on the contrary, the rotation scores of PD rats in the Control group kept stable at these observational points, which showed a significant statistical differences between Transplantation and Control group (P<0.05).4. Most of the grafted cells were Brdu/DAT double positiveHistochemical analyses showed that most of grafted cells made a positive expression of special markers of dopamine neuron in the treated rats:tyrosine hydroxylase and dopamine transporter5. The grafted cells displayed a potential of releasing dopamine neurotransmitterThe ELIS A results established that dopamine in the supernatants of transplantation group was up to (1954.12+14.79)pg/g, however, the dopamine in the supernatants of control group is (1687.94±14.82)pg/g. The transplantation group is obviously higher than the control group, P<0.05.Conclusions:1. It has been established that PB-IPC make an apparent expression of Sox2, Nanog, c-Myc, Klf4, Ngn1, Ngn2and Mashl.2. PB-IPC could differentiate into mature dopamine neurons. Transplantation of PB-IPC could make an obvious therapeutic effect on PD rats. |
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