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ABSTRACT The carapace of the tortoise Chinemys reevesii is an ingredient of "Guijia", a traditional Chinese medicine. However, C. reevesii is difficult to raise in aquaculture and is rare in the wild. Counterfeit tablets are made from carapaces of other species. In addition to C. reevesii, other species including Mauremys sinensis, Indotestudo elongate and Trachemys scripta have been used in Plastrum Testudinis as well. After processing, these carapaces are difficult to identify on the basis of morphological characteristics, which impedes law enforcement. Our study used DNA barcoding technology to identify C. reevesii and its substitutes. We extracted concentrated genomic DNA for PCR amplification. Based on the analysis of 61 full-length COI sequences, we designed four pairs of mini-barcode primers: Tu-A, Tu-B, Tu-C and Tu-D. The Tu-B primers sequenced genomic DNA with a success rate of 76.47%, and the Tu-D primers sequenced genomic DNA with a success rate of 88.24%. The identification efficiency of these two mini-barcodes was 70.59% and 64.71%, and the overall identification efficiency was approximately 76.47%. Similarly, a set of mini barcode systems was generated, which may provide an effective and low-cost method for the identification of authentic tortoise shells.
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Objective To explore the effect and the mechanism of vitamin D(Vit D) promotes proliferation and differentiation of mesenchymal stem cells (MSCs) through regulates extracts of plastrum testudinis (PTE).Methods Established the PGL3-Id1 promoter and transfered rat MSCs.PTE combined with 10-6,10-7,10-8mol/L Vit D respectively were acted on the transfected MSCs for 36 hours.The level of Id1 promoter were detected by luciferase activity measurement.1,3,30,100 pg/mL PTE combined with Vit D of 10-7 mol/L were acted on MSCs for 36 hours,3 days and 7 days,and the VDR expression were detected by RT-PCR test.Results PTE promoted the expression of Id1 in MSCs,the expression of Id1 was inhibited when PTE combined with Vit D (P ＜ 0.01),and it was significantly different among different dosis of Vit D(P ＜0.01).The expression of VDR was inhibited in different degree when PTE combined with Vit D for 36 hours,3 days and 7 days.PTE combed with large dose of Vit D for 36 hours had significant effect of inhibition,and the difference was statistically significant (P ＜ 0.05).The inhibiting effect was more obvious when PTE combined with large dose of Vit D for 3 days and 7 days.When different doses of PTE combined with Vit D for a same duration,the difference of VDR expression was statistically significant (P ＜ 0.05).Meanwhile,when same doses of PTE combined with Vit D for different durations,the difference of VDR expression at 7 days and 36 hours was statistically significant (P ＜ 0.05).Conclusion The proliferation of MSCs which promoted by PTE was inhibited by Vit D,and the nuclear receptor VDR may be one of the targets of drug action for PTE regulating proliferation and differentiation of MSCs.
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Objective To explore the changes of microRNA expression during the neuronal differentiation of neural stem cells (NSCs) induced by Plastrum testudinis extract (PTE) . Methods NSCs were isolated from the hippocampus of 14-day SD rat fetus and were obtained after primary culture. The abilities of NSCs differenting into neurons were identified by immunofluorescent staining. NSCs were randomly divided into three groups, blank control group, and low- and high-dose PTE groups ( 3, 30 μg/mL PTE) . After the cells were incubated with PTE for 7 days, total RNA was isolated and then the expression of miR-124 and miR-9 was observed by real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR) . Results Compared with the blank control group, the expression of miR-124 and miR-9 remained unchanged in low-dose PTE group, but was significantly increased in high-dose PTE group ( P<0.05). Conclusion PTE promotes NSCs differentiating into neurons, which might be associated with the up-regulation of miR-124 and miR-9.
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OBJECTIVE:To discuss the improvement in Soxhlet extraction with ore and shell hard Chinese meteria madica taking Plastrum Testudinis as example.METHODS:On the basis of normal Soxhlet extracter,larger Soxhlet extracter on Plastrum Testudinis was experimented using saturated salt-water-bath,quadruplicity cover circle and timer.Large Soxhlet extracter was compared with normal Soxhlet extracter and stirring-reflux method by HPLC analysis,yield and clearance rate of DPPH.RESULTS:There were no significant difference between normal and larger Soxhlet extracters,but the stirring-reflux method was inferior to these two methods.CONCLUSION:The improved larger Soxhlet extraction is a thorough,high-efficient,convenient,economical and healthy extracting method for ore and shell hard Chinese materia medica.
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[Objective] To observe the effect of serum containing Carapax et Plastrum Testudinis (CPT) in inducing the differentiation of mesenchymal stem cells (MSC) of adult rats into neurons. [Methods] The fifth generation of MSC were induced by the serum containing CPT (sero-CPT) and the expressions of neurofilament (NF) protein and glial fibrillary acidic protein (GFAP), the marks of neuron-like cells were detected by immunohistochemical method. [Results] The expression of NF protein was positive after the stimulation of sero-CPT and the expression reached the peak 12 hours after induction. [Conclusion] Sero-CPT can induce the differentiation of MSC into neurons in vitro.
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Objective: To explore the effect of Plastrum Testudinis on the three subtypes of NOS (nNOS,iNOS,eNOS) expression in rats with focal cerebral ischemia. Methods:Rat model of focal cerebral ischemia was set up by inserting nylon thread into the internal carotid artery . The expressions of nNOS, iNOS and eNOS was detected by immunohistochemical method to observe the effects of Plastrum Testudinis. Results: Expressions of nNOS and iNOS in cerebral cortex and caudate-putamen of Plastrum Testudinis treatment group were significantly lower than those of model control group (P
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Objective To investigate the effect of Plastrum Testudinis on neural stem cell after focal cerebral ischemia.Methods Rat models of focal cerebral ischemia were established by inserting nylon thread into the anterior cerebral artery.Immunohistochemical method was used to detect the expression of the neuroepithelial stem cell protein(Nestin),neurologic deficit and histological features were also observed.Results Plastrum Testudinis can decrease the neurologic dificit score and promote the expression of Nestin.Conclusion Plastrum Testudinis can alleviate the neurological symptoms and promote the proliferation of neural stem cell after focal cerebral ischemia.
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[Objective] To observe the effect of extract of Carapax et Plastrum Testudinis (ECPT) on nuclear receptor in the proliferation of rat mesenchymal stem cell (MSC) in vitro. [Methods] The rat MSC dissociated from bone marrow by density gradient method were cultured and identified by marking of bromodeoxyuridine (Brdu) and staining of CD44. Then different doses of ECPT (3333, 333.3 and 33.33 ?g/mL) were respectively added into in-vitro cultured MSC for 12, 24, 72 and 120 hours. The expression of retinoic acid receptor-?(RAR?), vitamin D receptor (VDR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor-?(TR?) and peroxisome proliferator-activated receptor ?(PPAR?) in MSC was detected by immunohistochemistry and immunofluorescence methods. [Results] The number of RAR?-and VDR-positive cells in ECPT groups was higher than that in the control group (P
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Objective To compare the effects of two crushing methods on dissolution rate and decocting rate of water-soluble protein in Plastrum Testudinis.Methods The dissolution rate of water-soluble protein was compared in Plastrum Testudinis processed by the traditional crushing method(passing 100-eyes screen,fine powder)and ultra-fine crushing method(passing 300-eye screen,ultra-fine powder);besides,orthogonal design was applied to study the process technical condition of decocting rate in Plastrum Testudinis.Results Water-soluble protein in fine powder of Plastrum Testudinis was hardly detected while the dissolution rate of water-soluble protein in ultra-fine powder was 51.2 %in 20 minutes and up to 70.5 %at 2 hours.Three influencing factors of fineness,decocting frequencies and decocting time were measured with orthogonal test.The results of variance analysis showed that fineness significantly influenced the decocting rate(P .05).Conclusion The ultra-fine powder technique is propitious to the increase of dissolution rate and decocting rate of water-soluble protein in Plastrum Testudinis.
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Objective To investigate the effects of kidney-tonifying Plastrum Testudinis on o steogenesis of cultured rat mesenchymal stem cells(MSCs ).Methods MSCs were isolated from adult rats wi th density gradient separation meth od.Osteogenesis of MSCs in the culture a dded with different concentrations of serum containing Plastrum Testudinis was evalu-ated by detecting bone glaprotein(BGP)level and observing the morphologic al features of MSCs and by alkaline ph os-phatase(ALP)and Von Kossa immunohistochemical m ethods.Results Morphological examination showed t hat the MSCs attachment formed soon after seedin g ,and grew into colonies with the appearance of fibroblastic cells.Seru m containing Plastrum Testudinis promoted the expression of alkaline phosphatase(ALP)in MSCs ,and increased BGP level and the number of calcified deposition in dose -dependant manner,the difference being significant in comparison wi th the control groups(P