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Background About one third of congenital cataract is associated with inheriting factor.The inherited heterogeneity has been found in congenital cataract.To seek the pathogenic gene is essential for the gene therapy. Objective Present study was to map and identify the causal gene for autosomal dominant congenital cataract (ADCC) in a Chinese family. Methods The clinical features of all affected members in this family were examined.Blood samples were collected from eleven family members for genetic linkage analysis.Polymorphic microsatellite markers were selected from the regions which harbor all known loci linked with ADCC.Universal fluorescent-labeled M13 primer was used in linkage analysis.Direct genomic sequencing was used to evaluate the candidate gene for example CRYBB2 gene.This study followed Helsinki Declaration and was proved by Tianjin City Ethic Committee.Written informed consent was obtained from each SUbject before any medical procees. Results The maximum two-point LOD score of 1.20 was obtained for marker D22S315 (θ=0).The LOD score of 0.6 was obtained for marker D16S3068.No mutation in all exons of CRYBB2 gene was found in the family. Conclusion CRYBB2 gene associated with ADCC was excluded from the family.A genome-wide linkage screening should be conducted.Genotyping with microsatellite markers using Universal fluorescent-labeled M13 primer can decrease the cost and obtain the same result.
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AIM: To investigate the CdTe quantum dots coated with MPA and explore its biocompatibility with living cells. METHODS: CdTe quantum dots coated with MPA were prepared in aqueous phase and MPA CdTe QDs were Characterized with TEM, fluorospectrophotometer and ultraviolet spectrophotometer. QDs were Modified with with avidin, purified and prepared as flurescent probe. LSCM was used to observe the expression of MHC Ⅱ antigen on PMφ cells, which was labeled by QDs. Cell culture and MTT assays were used to determine the biocompatibility of MPA coated CdTe quantum dots with the B-16 cells as target cells. RESULTS: The particle diameter of CdTe quantum dots prepared in aqueous phase was well distributed. They had good photological performance and greater stability after coated with MPA. MHC Ⅱ antigen on PMφ was labeled with the QDs-Avidin fluorescent probe showed great fluorescence intensity, which was easy to be detected by fluorescence microscope and LSCM. MPA CdTe QDs showed cytotoxicity when its density was very high, but they showed little cytotoxicity during the normal use of influence label density limit. CONCLUSION: MPA CdTe QDs can be used as new fluorescent lable as they are of even size, not easy to bleach or quench, have good photological performance and stability and good biocompatibility.
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Objective To study the double restriction fluorescent labeling (DRFL) method for fluorescent labeling of trace DNA samples and its effect in enhancing the pathogen detection sensitivity of microarray assays. Method SARS-CoV RNA samples were reversely transcribed and then further amplified with the restriction display (RD)-PCR and fluorescently labeled by conventional restriction labeling directly with Cy-universal primer and the novel double labeling with Cy-universal primer and CydNTP. The labeled samples were applied to the microarray with the viral probes, processed and analyzed. Results Compared with the conventional method, DRFL labeling resulted in 3. 5835 times higher fluorescent intensity of all the SARS probes on average, even though increased fluorescent intensities for different probes varied considerably. Conclusion Signal to noise ratio can be enhanced by the DRFL method which improves the sensitivity of microarray technology in trace pathogen detections.
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Objective To explore the effect for nerve regeneration with immunosuppresssant FK506 a-round the cut after end-to-side neurorrhaphy of peripheral nerve. Methods There were 40 adult SD rats and were divided into two groups randomly by means. In the experiment group, the right peroneal nerve of SD rat was cut off,then an 1mm epineural window was created on the neighboring tibial nerve,the distal end of peroneal nerve was sutured to the windowed tibial nerve by means of end-to-side attachment, application of slow-releasing diaphragm which was implanted around the nerve anastomosis with FK506. In the contrast group,there were not the FK506 around the cut after end-to-side neurorrhaphy. The tibial and peroneal nerve trunks were labeled by being in jected with fast blue ( KB) and fluorescence golden ( FG) respectively. The labeled cells in the dorsal root ganglia (DRG) and spinal cord were observed by fluorescence microscopy. Results There were a lot of FB labeled cells in the experiment group than in the contrast group in the DRG and spinal cord. Conclusion Immunosuppresssant FK506 could promote nerve regenerative speed and quality after end-to-side neurorrhaphy of peripheral nerve.
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Objective:To study the new bone formation after distraction osteogenesis (DO) to repair cleft palate (CP) by laser scanning confocal microscope (LSCM) and quantitive image analysis. Methods: 20 cats were assigned randomly to 3 groups: Experimental group (15 cats), the CP bone defects in hard palate were corrected with DO repair at the rate of 0.4 mm twice a day, till the transport disc (TD) reached the opposite edge across the defect region. The tetracycline labeling (30 mg/kg) were intramuscularly injected 6 days before specimens retrieval of each 3 cats at 2, 4, 6, 8 and 12 weeks after the completion of distraction. The sections were observed and analyzed by LSCM and quantitive image analysis. The results were then compared with empty control (2 cats) and experimental control group (3 cats). Results: The data showed that active new bone formation were observed in 2 weeks and reached the summit in 4 weeks. The bone formation slowed down through 8 to 12 weeks but still significantly stronger than empty and experimental control groups in which no new bone growth were observed. Conclusion:The hard palate bone defect is eventually reconstructed by DO repair and the distraction gap was filled with active de nove intramembranous osteogenesis. A consecutive dynamic curve of fluorescent labeling deposition implies active bone formation and remodeling is revealed.
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21 albino rats were used. The origin of the noradrenergic (NA) fibers in the septum was studied with a simultaneously combined retrograde fluorescent tracing and histofluorescence method. The results revealed that the NA neurons projecting to the septum were in A_1, A_2 and A_6 groups. The projections of A_1 were bilateral, But A_2 and A_6 projected dominantly to the ipsilateral septum, with a few to the contralateral. Retrograde labeled NA cells were localized in the rostrodorsal part of A_6 group, and scattered over the whole rostrocaudal length of A_(1,2) groups. No labeling was seen in A_4 and A_7 groups. In five of the animals, retrograde labeled NA cells were occasionally observed in A_5 group. In addition, some scattered labeled non-NA neurons were seen in the raphe and the ventrolateral reticular formation of the rostral medulla.
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Electrophysiological studies have shown that the spinosolitary tract-dorsal column postsynaptic (SST-DCPS) neurons may project to both the dorsal column and solitary tract nuclei. In order to demonstrate the neurons morphologically, fluorescein dyes, PI and Bb, were injected into the dorsal column nuclei and the solitary tract nucleus respectively. A total 282 cells were found to be retrogradely labeled in the spinal dorsal horn in 10 adult rats. Of them, 51 (18%) cells were PI-Bb doubly labeled; 120 (43%) were PI labled alone; and 111 (39%) were Bb singly labled. Most of these double-labled cells were concentrated in laminae III-V. The existence of double projection neurons that project to both the dorsal column and solitary tract nuclei, namely the physiologically identified SST-DCPS neurons, is morphologically confirmed in the present study. These neurons may transmit information to both visceral and somatic sensory nuclei, indicating they may play an important role in the convergence of somato-visceral afferents.
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Studies on the origin of the afferent acetylcholinesterase-containing fibres of the nucleus accumbens in the brain stem of 27 rats were carried out by retrograde fluorescent labeling combined with AChE histochemistry. DAPI or Pr was injected into the nucleus accumbens. The animals survived for 2.5-3 days. DFP was injected intramuscularly 6 to 8 hours before left intraventricular perfusion by 10％ formalin-containing phosphate buffer solution (0.1mol/L, pH 7.4). The frozen serial transverse sections of the brain stem were incubated in Karnovsky-Roots solution. The fluorescent retrograde labeled cells with AChE reaction-positive products were traced under OLYMPUS fluorescence microscope and light microscope simultaneously. The results were as follows: (1) On the ipsilateral side, the retrograde fluorescent labeled cells with AChE reaction-positive products were found in the following nuclei: area ventralis tegmentalis (AVT), substantia nigra (SN), nucleus raphes mesencephali caudalis(cmr), nucleus tegmenti pedunculopontinus(tpt), nucleus raphe dorsalis(dr), nucleus centralis superior (cs), nucleus reticularis tegmenti pontis(rtp), Locus ceruleus (Lc), nucleus tractus solitarus(tsol), nucleus raphe pallidus (rpa), nucleus reticularis paramedianus (rpm), subnucleus reticularis ventralis(rv), nucleus reticularis lateralis(rL). (2) On the contralateral side, a few of the above mentnioned cells were also found in AVT, SN, dr, cs, and tsol.