Objective To study the condition and method of hydrodynamics-based transgene(HDT) in rat fatty liver. Methods Inject different dosages and concentrations of green fluorescent protein plasmid pEGFP-C1 at different speeds, then collect 4 rats' liver leaves at different time points after injection and prepare their frozen section, finally observe and quantify the GFP expression with fluo rescence microscope at 488 nm excitation wavelength. Results Plasmid pEGFP-C1 concentration 33mg/L, injection speed 2ml/s, injection volume 8.5%of rat body weight, injected plasmid. After 6 hours of injection, GFP-positive cells rate of pedicel leaf is about 18%, left leaf about 14%, middle leaf about 12.5%;R3ight leaf about 10% and tail leaf about 8%. GFP begin to gradually reduce since 24 hours, until 72 hours almost no GFP-positive cells were checked in all liver leaves. Conclusion Hydrodynamics-based transgene can be applied to rat fatty liver, the appropriate conditions of this method are 33mg/L plasmid concentration, 8.5% rat avoirdupois, 2ml/s injection speed, and the suitable time to observe the proportion of GFP-positive cells is 6-24 hours after gene injection.

Objective Measuring the displacement and deformation of the fifth screw under different tensile force before or after adultal internal fixation with compressing steel plate for femoral fractuer by digital speckle correlation method.And analysis of the displacement and deformation after reducing the number of screws.Aimed at providing a theoretical basis for clinical femoral internal fixation with compressing steel plate about the number of screws. Methods Taking the antisepsis femoral specimen of grown-ups, embedding and fixing with tooth powder after removing the soft tissue, producing a fixed model in the 1/2 femur.Fixing steel plate below the lateral femur subperiosteal with five screws through the fourth floor of cortical bone on both sides of the fractures, samples were divided into 10 states, group A-J were measured by orders, under the rally of 400 N,500N , measuring the displayment of the fifth screw with CSS-44100-e-universal test machine for loading rally experiment ,and automatic data processing. Results For analysis of variance, the mean displacement measurements of the fifth screw under rally of 400N,500N, B-J group and A group have significant differences(P<0.05). The mean deformation measurements of the fifth screw under rally of 400N,500N, group A and group F-J group have significant differences(P<0.05);group A and B-E group have no significant differences.Conclusion In a certain range, the fracture displacement and the numuber of the screws show negative correlation. The screws near the fracture line for femoral compression plate internal fixation of femoral fractuer is easily broken because of the too large deformation.

Objective Using shotgun mass spectrometry to detect proteins probably contained in bone marrow stromal cells(BMSCs) conditioned medium. MethodsMixed with BMSCs conditioned medium was divided into two parts which is(>5kD and <5kD) by means of ultrafiltration. The two parts were used to culture neural stem cells(NSCs) separately, and the proportions of neurons, astrocytes and oligodendrocytes in the offsprings of NSCs were calculated, then the effective part that could regulate the differentiation of NSCs was detected by Shotgun mass spectrometry. Results The BMSCs conditioned medium which is >5kD could promote the NSCs differentiate into more neurons and oligodendrocytes. The SDS-PAGE of this part showed that the most proteins were above 14kD, then the protein bands were enzymed. In total, 456 proteins were identified by Shotgun mass spectrometry after all the protein bands were enzymed, there were 154 similar proteins, 17 hypothetical proteins and 56 unknown proteins. And in the rest of 229 proteins, most of them were cytoskeletal proteins, secreted proteins, signal transduction proteins, enzymes, transporter and so on. Conclusion Many proteins secreted by BMSCs could regulate the differentiation of NSCs so as to prove the protein components probably existed in the BMSCs conditioned medium.

Objective To investigate the influence of pinealectomy(Px)and melatonin (MLT) supplementation on CD4~+CD25~+Treg cells development and Foxp3 expression in rat thymus. Methods One hundred and twenty clean-grade male SD rats were divided into five groups: normal control group, sham pinealectomized group, pinealectomized group, Px +7.5 mg/(kg·d) MLT group, Px +15 mg/(kg·d) MLT group, and the thymus were taken out at the 4th week and the 8th week;Flow cytometric analysis was used to analyse the number of double positive cells in the thymus and peripheral blood;Semiquantitative RT-PCR and immunohistochemistry were applied to analyse Foxp3 expression of rat thymus. Results There was no difference of the number of CD4~+CD25~+ Treg cells in rat thymus between Px and normal/sham group, and the number increased dependently on time and dose after MLT supplementation. In rat peripheral blood the double positive cells significantly increased in models of Px at the 4th week, and then backed to normal level after MLT supplementation. The results showed no significant changes in all groups at the 8th weeek;At the 4th weeks, Foxp3 expression increased in the thymus of Px rats compared to nomal/sham group, which returned to normal level after MLT supplementation. On the other hand, Foxp3 expression showed no significant difference in all groups at the 8th weeek.Conclusion Px made no difference between the development of CD4~+CD25~+Treg cells in rat thymus, but resulted in significant increase of thymic output and Foxp3 expression. All the effects disappeared at the 8th week. MLT supplementation could reverse the abnormity.

Objective To observe how exogenous estrogen influences the distribution and the expression of NOS positive neurons in the supraoptic nuclei of hypothalamus in the overiectomized female rats. Methods The 2-3-month-old female rats(n=40) were selected as the healthy and nulli-copulatory experimental animals. Rats were divided into following groups: normal control group(n=10), ovariectomized control group(n=10), and two experimental groups that have been injected with estrogen for post-operative 40 days(n=10) and for post-operative 70 days(n=10). Finally, all the animals were infused and the brains were removed. Immunohistochemical (SABC) method was adopted to count the number of NOS poitive neurons and observed the NOS poitive neuronal morphology under the light microscope. The image analysis system was used to test the average gray value of immunoreactivity in NOS positive neurons. Results In the ovariectomized control group, the density of NOS positive neurons in supraoptic nucleus was significantly increased and their shapes were bigger than that of the normal control group(P<0.05). The density and the form of the NOS positive neurons in supraoptic nucleons had no apparent difference between the estrogen for post-operative 40 days group and the ovariectomizeed control group(P>0.05).In the group after estrogen-injection 2 months compared with the normal control group, and the ovariectomized control group, both of the NOS positive neurons' density and the size become significantly decreased, and the staining of cells was lesser in the group injected with estrogen for post-operation 70 days. Conclusion The present results suggest that exogenous estrogen may influence the distribution and the expression of NOS positive neurons in supraoptic nucleus of hypothalamus of ovariectomized female rats.

Objective To explore the effect and injury mechanism of reactive oxygen species (ROS) after spinal cord injury (SCI) through detecting the dynamic changes of malonyldialdehyed (MDA)content in spinal cord and observing neurocyte apoptosis and correlation apoptosis factor expression after SCI. Methods Totally 132 adult SD male rats were randomly divided into three groups: sham group, SCI group, methylprednisolone (MPSS) group. The SCI of SD rats was performed by Allen's weight dropping way to impact on the posteriors of spinal cord T_(10). The contents of MDA were determined by chromatometry, the expression of Caspase-3 and Bcl-2 family in the injured spinal cord was detected by immunohistochemical staining;Apoptotic cells were detected by using fluorometric terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (fluorometric TUNEL) staining. Results The content of MDA in the injured cord increased significantly after SCI;R3eached the peak at 6 hours and 3 days post-injury, then dropped down gradually, then was back to the normal level after 7 days. The number of TUNEL labeling positive cells of SCI group increased at 6 hours post-injury;R3eached the peak at 3 days, then dropped down gradually;Bcl-2, Bax protein began to increase at 6 hours post-injury;R3eached the peak at 5 days after injury, then dropped down gradually. Caspase-3 protein began to increase at 6 hours post-injury;R3eached the peak at 3 days after injury, then dropped down gradually. The content of MDA, the number of TUNEL labeling positive cells, the expression of Caspase-3 and Bax of MPSS group decreased significantly than that of SCI group at the same time;R3espectively, while Bcl-2 protein was up-regulated after administration of MPSS.Conclusion ROS could promote the expression of Caspase-3 and degrade the ratio of Bcl-2/Bax to induce apoptosis of neurocyte, which might play significantly role in the process of secondary SCI. In addition, MPSS exerts neuroprotective effects against ROS toxicity, which might be of importance and might contribute to their clinical efficacy for the treatment of SCI.

Objective To investigate the neuroprotective effects of picrodideⅡ on cerebral ischemic reperfusion injury in rats. Methods Intraluminal thread methods were applied to establish the left middle cerebral artery occlusion reperfusion models (MCAO/R) in rats. PicrodideⅡ (10mg/kg) and salvianic acid A sodium (10mg/kg) were injected from tail vein for treatment. The neurological behavioral function was evaluated with Bederson's test. The cerebral infarction volume was observed with tetrazolium chloride (TTC) staining. The structure of cells was observed with histopathology. The apoptosis positive cells were counted by terminal deoxynucleotidyl transferase midiated dUTP nick end labeling (TUNEL). Results The neurological behavioral malfunction appeared in all rats with MCAO/R. The infarction focus showed in the ischemic hemisphere following cerebral ischemia reperfusion injury. In the picrodideⅡ and salvianic acid A sodium treatment groups, the number of apoptosis positive cells decreased and the cerebral infarction volume reduced, while the neurological behavioral function was significantly improved than those in the model control group (P<0.05). The cerebral infarction volume in the picrodideⅡ group was smaller than that in the salvianic acid A sodium group (P<0.05).Conclusion PicrodideⅡ might reduce cerebral infarction volume and improve the neurological behavioral function through inhibiting the neuronal apoptosis induced by ischemia reperfusion injury.

Objective Kunming strain(KM) mice were used as animal models. Nontoxic dextran conjugated with tetramethylrhodamine(TMR)and fluorecein isothiocyante(FITC)was microinjected to two of the 2-cell blastomere as molecular probe to trace the development fate of the blastomere ,in order to figure out the mechanisms of the formation of Em-Ab axis. Methods FITC- dextran was injected to zygote in order to make sure if it is noxious. Two blastomeres of 2-cell embryo were injected FITC- dextran and TMR- dextran respectively. Results When labeled embryo develeped to blastocyst, distribution of progeny of 2-cell embryo blastomeres can be detected.Conclusion The cells of blastomere randomly distributed either embryonic parts or extraembryonic parts of blastocyst.

Objective Revealing the developmental regulation of Cervus nippon's oocyte and organelles. Methods In the experiment,follicle systems during both estrum and non- estrum were divided into the primordial follicle,growing follicle and mature follicle according to the Cervus nippon's follicle diameter size,formation of zonapellucida,appearance time of follicular cavity.At the same time,observations on cytoplasmic organelles in development of oocytes were conducted with electron microscopic,eyepiece micrometer and photomicrographic technique(The number of every oocyte observed is 6-8). Results In the primordial follicle and early growing follicle phase,the quantity of mitochondria,golgi apparatus,smooth endoplasmic reticulum and cortical granules increased gradually and all organelles moved to the cortical area.However,in the late growing follicle and mature follicle phase,Golgi apparatus and rough endoplasmic reticulum disappeared,cortical granules began to arrange themselves in line beneath the plasma membrane of the oocyte,mitochondrias dispersed toward the central region of cytoplasm,and almost all the round mitochondria with rare cristae turned into hooded ones, and nucleus compaction occurred.In addition,the short and thick microvilli began to appear from the primary follicle ovocyte,become intensive and slender when secondary follicle's ovocyte;It's until tertiary follicle's ovocyte,microvilli started to shorten and become coarse,and even parts of them contract from the zona pellucida gradually.Conclusion In the development of oocytes, the changes of type,quantity and distribution on mitochondria has a close relation with cells at proliferation,differentiation and metabolism level.Cortical granule has no association with golgi apparatus basically,but smooth endoplasmic reticulum(SER).The nuclei are the sites of RNA synthesis and warehouses,and its densification is the premise of meiosis recovery.

Objective To investigate the development and prognosis of peroxisome proliferator-activated recepor-γ(PPAR-γ) and cyclooxygenase-2(COX-2) in endometrial carcinoma. Methods The electron microscopic immunohistochemical technique was used to observe the ultrastructure location of COX-2 protein labeled by colloidal gold in the endometrial carcinoma. Results 1.There were negative immunohistochemical staining signals of PPAR-γ in both the normal endometrium and hyperplasia endometrium, whereas, was positive staining in the endometrial carcinoma;2.The intensities of the COX-2 immunohistochemical staining were significantly statistical difference among the normal endometrium, the hyperplasia endometriu and the endometrial carcinoma(P<0.01);3. The COX-2 labelled by colloidal gold granules was observed in the endoplasmic reticulum, nuclear membrane and nuclei in the endometrial carcinoma. Conclusion Both PPAR-γ and the COX-2 might play an important role in the development of the endometrial carcinoma.