Objective To explore the feasibil ity of using PKH26 as a cell tracer to construct tissue engineered bone. Methods BMSCs isolated from the bone marrow of 1-week-old New Zealand white rabbit were cultured. The BMSCs at passage 3 were labeled with PKH26 and were observed under fluorescence microscope. The percentage of the labeled cells wasdetected by Flow cytometer. The labeled cells were induced to differentiate into osteoblasts in vitro and the morphology of the cells after induction was observed under inverted phase contrast microscope. The osteogenic induction was evaluated by ALP staining and Alizarin red staining. The cells labeled with PKH26 were seeded on the bio-derived bone to construct tissue engineered bone in vitro. Then the compound of cells and material were observed under fluorescence microscope. The compound of labeled cells and material were implanted into the rabbit thigh muscle, and the transformation of the labeled cells was observed by fluorescence microscope 14 and 28 days later. Results Fluorescence microscope observation: the BMSCs labeled by PKH26 were spherical and presented with red and uniform-distributed fluorescence, and the contour of the cells were clearly observed when they were adherent 24 hours after culture. Flow cytometric detection revealed that the percentage of labeled cells was 97.2%. After osteogenic induction, the morphology of the cells changed from long-fusiform to polygon-shape or cube-shape, more ECM was secreted, andthe ALP and the Alizarin red staining were positive. At 48 hours after culturing the PKH26 labeled BMSCs with bio-derived bone, the fluorescence microscope observation showed that there was red fluorescence on the surface and inside of the material. At 14 days after implantation, the labeled cells with red and l ight fluorescence were evident in the implantation area; while at 28 days, the cells with red fluorescence were still evident but less in quantity and weaker in fluorescence strength. Conclusion PKH26 can be used as BMSCs label for the construction of tissue engineered bone in vitro and the short-term tracing in vivo.
Objective To investigate the venous drainage in retrograde island flaps by fluorescence tracing technique and to observe the pathway of venous drainage. Methods The 0.1mL venous blood was collected from the marginal ear vein of every rabbit (n=20), respectively, and erythrocytes were separated by centrifugation and then were labeled with FITC. Positive rate and fluorescence intensity of FITC-labeled RBC were detected by flow cytometry. RBC morphous was observed under the inverted fluorescence microscope. Saphenous retrograde island fasciocutaneous flap and antegrade islandfasciocutaneous flap (4.0 cm × 3.0 cm in size with vascular pedicle length of 3.0 cm) were successfully establ ished in hind l imbs of 20 New Zealand white rabbits．One hind l imb of each rabbit was randomly assigned as the experimental group and the contralateral side was assigned as the control. The same flap was establ ished in the control group without any fluorescence tracer. According to retrograde or antegrade flaps, the experimental group was divided into 2 groups with 10 rabbits in each group. And then, according to different pathways of tracer-giving, each group was divided into 2 subgroups of artery and vein, with 5 rabbits in each subgroup. The labeled erythrocytes (5 μL) were injected into artery or vein and then flaps were cut down 5 seconds later. The flaps were immediately frozen and chipped (5-7 μm). Consecutive three frozen sections were made and two of them were stained with HE and GENMED, respectively, but the third one was squashed without staining. All frozen sections were observed under the microscope. Results Positive rate of FITC-labeled RBC was beyond 99% and fluorescence intensity was more than or equal to 103. FITC-labeled RBC showed steady green fluorescence under the inverted fluorescence microscope. Fluorescence appeared in all experimental groups, but none was found in the control groups. In antegrade island flap group, fluorescence appeared mainly in lumen of vein, wall of vein and inner membrane and outer membrane of artery. In retrograde island flap group, fluorescence distributed principally in inner membrane and outer membrane of artery and wall of vein. Conclusion The fluorescence tracing is appl icable to the research of venous drainage. Venous drainage in the antegrade island flaps is mainly through lumen of vein, wall of vein and inner membrane and outer membrane of artery. While, venous drainage in retrograde island flaps is principally through inner membrane and outer membrane of artery and wall of vein.
Objective To establish a kind of gene therapy method of rheumatoid arthritis, to construct the interleukin-18-PE38 fusion gene expression vectorand to explore the expression of the fusion gene in the chondrocytes and 3T3 cells. Methods Interleukin-18-PE38 fusion gene was cleaved from plasmid PRKL459k-IL-18-PE38 by restriction enzyme digestion,then linked with vectors PsecTag2B and transformed into competence bacteria, positive clones were selected and confimed by restrictive enzyme(EcoRI) digestion assay. The rearrangement plasmid PsecTag2B-IL-18-PE38 was transfected into 3T3 cells and mouse chondrocytes by liposome protocol(experimental group),null vector was used as negative control, and the transient expression was identified by fluorescence immunocytochemical assay. Results Restrictive enzymes digestion analysis revealed thatthe length of theinterleukin-18-PE38 fusion gene was 6 000 bp. Fluorescence immunocytochemical method showed that fluorescence intensity of the experimental group is b,whilefluorescence intensity of the control group is weak. Conclusion the eukaryoticexpression vector PsecTag2B-IL-18-PE38 is established successfully which canbeexpressed in the 3T3 cells and mouse chodrocytes. Our results lay a foundationfor the further investigation for rheumatoid arthritis therapy.
Objective To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons.Methods In the 4thgeneration of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342(Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguishthem. The seeding cells were collected to make them to be the cell suspension of suitable concentration(6.0×105 cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and didnot cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to noncryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreservedcell to non-cryopreserved cell and the cell survival rates. We compared the cll survival rates between immediate flow cytometry and that 2 hours after fluorescence staining. Results The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r=0.970，Plt;0.05), image analysis study also showed the correlation was significant (r=0.982，Plt;0.05).The cell survival rate decreased by use of flow cytometry twohours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (Pgt;0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly.Conclusion Flow cytometry and fluorescence image afterPI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservationof tissue engineered tendons.
Objective To observe the image characteristics of autofluorescence (AF) in central serous chorioretinopathy (CSC). Methods A total of 85 eyes of 72 patients with CSC were examined by Headberg HRA2 laser scanning fundus fluorescein angiography (FFA), redfree light photography, and Kowa fundus colorizedphotography. The grey AF images were obtained with 488 nmwave-length laser and comparatively analyzed with results of fundus colorized photography, redfree light photography and FFA. Results In 85 eyes, single faint AF of the CSC focus was in 14 (16.5%); faint AF pool containing b lamellar focus was in 39 (45.9%); faint AF pool combining with mottling focus was in 25 (29.4%); local dense or scattered mottling AF at the posterior pole was in 7 (8.2%). FFA fluorescein leakage point or abnormal fluorescence were in accordance with abnormal AF in 60 eyes (70.6%); the changes of ocular fundus, results of FFA, and changes of AF were not accordance in 25 eyes (29.4%). AF of CSC focus during different disease course was different, which showed single platelike faint AF pool and b mottling AF complex focus in and out of the faint AF in the period of onset of the disease, while b mottling combining with faint mottling AF and various multiinfection fields in the period of chronicity. Conclusions The AF of CSC mainly demonstrates single faint AF, b mottling combining with faint mottling AF and multiinfection AF in macular fields. AF examination associates with fundus colorized photography and FFA can be mutually complemented in observing the images of CSC.
Objective To observe the autofluorescence of dated fundus hemorrhage excited by the excitaton light with different wavelength. Methods A total of 23 patients (23 eyes) with dated fundus hemorrhage were observed. The blue light under the fundus fluorescence angiography (FFA) mode of Topcon 50IA fundus camera was the excitation light, and the whiteandblack images of 4 patients and colorized images of 16 patients were collected, respectively. The autofluorescence of dated fundus hemorrhage in other 3 patients was observed by excitation of scanning laser with the wavelength of 488 nm and 795 nm emitted from Heidelberg retina angiography apparatus (HRA2). Results The black and white images showed the b red autofuorescence of dated fundus hemorrhage in 4 patients, while the colorized ones revealed the red autofluorescence in 16 patients. The hemorrhage autofluorescence could be also excited by blue laser (488 nm) and infrared laser (795 nm) using HRA2, but with different extent and intensity. Conclusions Due to the complex composition of dated fundus hemorrhage, different excitation light can excite the autofuorescence with different wavelength.
Objective To observe the pathological and functional changes of retinal photochemical damages exposed to green flurescent light. Methods The Sprague Dawley rats were continually exposed to green fluorescent light with an illuminancem level of (1 900plusmn;106.9) Lx for 24 hours.The changes of retinal morphology and morphometrics and flash electroretinogram were studied before light exposure and at the 6th hour,6th day and 14th day after light exposure. Results At the 6th hours after light exposure,the outer nuclear layer(ONL)of retina becoma thinner compared with that bfore light exposure.The thickness of ONL decreased by 23.91% and the inner and outer segments appeared disorderly arranged.At the 6th day after light exposure the thickness of ONL is thinner than that at the6th hour,i.e.decreased by 46.6%. At the 14th day after light exposure the thickness of ONL decreased by 42.40%.Flash electroretinogram showed that the amplitudes of a and b wave decreased continuously at the 6th hour and 6th day and unrecovered at the 14th day after light exposure. Conclusion This model might be an ideal one for research on retinal photochemical damage. (Chin J Ocul Fundus Dis,1998,14:101-103)
ObjectiveTo establish a more accurately method to detect the residue of 1,4-butanediol diglycidyl-ether (BDDE) in the cross-linked sodium hyaluronic gel so as to provide a scientific testing method for the quality control. MethodsThe gas chromatography was used to explore the thermal stability of BDDE, and the residues of BDDE in sodium hyaluronic gel was detected by fluorescence spectrophotometry. The hyaluronidase was added to the BDDE standard solution and the improved fluorescence spectrophotometer was used to detect the BDDE residues in the cross-linked hyaluronic sodium gel. ResultsA good linearity was obtained as y=14.102x+1.103 (R2=0.999 8) for BDDE. BDDE was unstable under high temperature and long storage time. The relevant fluorescence intensity was detected with hyaluronidase solution. After adding hyaluronidase into the BDDE standard solutions, the advanced linearity was obtained as y=14.027x+7.062 (R2=0.999 9). ConclusionFluorescent spectrophotometry is a simple, rapid, and accurate method to analyze BDDE residue of cross-linked sodium hyaluronic gel. Due to the poor thermal stability, all the factors related to temperature must be excluded during the process, including the temperature control of every step. Furthermore, the adding of hyaluronidase in the pre-preparation of cross-linked sodium hyaluronic gel can bring interference. So when using fluorescent spectrophotometry, adjustment must be taken before the calibration curve is preparation.
Autofluorescence is produced by lipofuscin in retinal pigment epithelium (RPE) cells which is induced by exciting light and enables the visualization of lipofuscin changes in the RPE cells, thus showing the function of RPE and photoreceptor cells. Fundus autofluorescence (FAF) imaging is a non-invasive imaging technique providing information of RPE and photoreceptor cells, which is not obtainable with other imaging modalities. The scope of applications includes identification of diseased RPE in retinal diseases, elucidating pathophysiological mechanisms, estimating disease progression and prognosis, guiding treatment protocols. Common fundus diseases have different pathological types, levels and causes, so they can cause various damages of RPE and photoreceptor cells which induce complicated FAF. It is worth to further observing and investigating the common retinal diseases' FAF characteristics and clinical applications.
Ultra-wide field fundus autofluorescence (FAF) imaging is a new noninvasive technique with an imaging range of about 200 °. It can detect peripheral retinal lesions that cannot be found in previous FAFs and more objectively reflect intracellular content and distribution of lipofuscin in the retinal pigment epithelium (RPE) and RPE cell metabolic status. The ultra-wide field FAF can find the abnormal autofluorescence (AF) in the peripheral retina of the eyes of age-related macular degeneration (AMD), and different AF manifestations may have an impact on the diagnosis and treatment of the different AMD subtypes. It is helpful to evaluate subretinal fluid in the eyes of central serous choroidal retinopathy and can accurately detect the changes in the outer retina of the eyes without subretinal fluid. It can help to determine the type of uveitis and fully display the evolution of the disease. It can also assess the peripheral photoreceptor cell layer and RPE in patients with retinal dystrophy and retinitis pigmentosa, and comprehensively evaluate their retinal function and monitor the progress of disease. It can also assist in the evaluation of the short-term efficacy and RPE cell function after the scleral buckling surgery for patients with rhegmatogenous retinal detachment. In the future, ultra-wide field FAF may change the knowledge and intervention strategy of ocular fundus diseases and promote the clinical and scientific research in this field.