Volume 39 ·  Issue 07 ·  2003

To determinate the existence of the inositol 1, 4, 5-triphosphate receptor (IP₃ R) and the ryanodine receptor (RyR), identify their isoforms and assess their functions during calcium ion concentration modulation in a human epithelial cell line (LEC-B3).

LEC-B3 cell line was defrosted and subcultured, the 3rd passage cells were chosen as object when they became confluent. Then the total cellular RNA was extracted from the cells, the expression of mRNA of IP₃R and RyR isoforms was analyzed by polymerase chain reaction. Ca²⁺ ion changes in the same passage cells were observed and analyzed with laser scanning confocal microscope when aceylcholine (Ach), atropine, caffeine, ryanodine, and procaine were added to the extracellular Ca²⁺, Mg²⁺ free solutions with ethyleneglycol bis ether tetracetic acid or Ca²⁺, Mg²⁺ involved solutions.

The IP₃R isoform I, III and RyR isoform I, III mRNA were expressed, the expression of isoform IP₃R III > IP₃R I (P<0.05); RyR III > RyR I (P <0.01). 10 μmol/L Ach, 10 mmol/L caffeine and 50 μmol/L ryanodine all could induce the intracellular free Ca²⁺ concentration increase, but the incubation periods, ranges and lasting time of the increases are different: when Ach acted, the duration of calcium increase in calcium involved solutions was a little longer than in calcium free solutions; the amplitude of calcium oscillations induced by Ach was much higher than caffein and ryanodine, while the onset of the later is slower than the former. Besides, 1 μmol/L atropine could block the Ach effects, 50 μmol/L procaine could inhibit the ryanodine effects.

(1) The IP₃R and RyR gene transcription coexist in the human lens epithelial cell line: LEC-B3, and their isoforms have specificity. (2) Different agonists can induce different calcium changes. (3) The increase of Ca²⁺ concentration induced by caffeine or ryanodine suggested the involvement of both the cyclic adenosine diphosphate ribose and the inositol 1,4,5-triphosphate systems in the rugulation of intracellular calcium in LEC-B3, and IP₃R is more sensitive to free Ca²⁺ modulation than RyR.

To detect the gene expression of Pax-6 in cultured lens epithelial cells (LECs) of mouse in vitro in order to study the gene factor that maintains the LECs characteristics.

Cultured the LECs (passage 1 to 3) were used in the study, the expression of Pax-6 gene in LECs was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemical staining and Western blot.

The mRNA of Pax-6 gene was positive in primary culture, the first and second passage of LECs using RT-PCR. The protein of Pax-6 gene was also positive in Western blot and immunohistochemical staining, but it was weak positive in the third passage of LECs and none in all passages of negative control groups.

The LECs are positive for the gene of Pax-6. The normal expression of Pax-6 gene is vital for maintaining the LECs characteristics.

To observe oxidative cataract inhibition of PD150606 by studying the opacity of the lens, the variation of protein and cells, and by further studying the role of calpains in the mechanism of oxidative cataract.

Rat lenses were cultured in vitro with H₂O₂ and PD150606. The lenses were observed under the microscope. Simultaneously, photograph analysis was performed in order to detect the variation of opacity; the proportion of WSP was determined by the method of Lowry; the number of apoptotic lens epithelial cells (LEC) was measured by flow cytometry; the expression of Caspase 3 in LEC was determined by SAS-PAGE electrophoresis and immol/Lunoblotting.

Opacity of lens was enhanced in 24 hours when cultured with H₂O₂ and PD150606. There were significant differences of relative gray scale between H₂O₂-induced and control group at 6, 12 and 24 hours (P<0.05), significant differences of the proportion of WSP between H₂O₂-induced and control group at 12 and 24 hours (P<0.01), significant differences of the number of apoptotic LEC at 6, 12 and 24 hours (P<0.01) and significant differences of expression of Caspase 3 at 6, 12 and 24 hours (P=0.000, 0.005, 0.004).

PD150606, an inhibitor of calpain, can inhibit H₂O₂-induced opacity of lens, proteolysis and apoptosis of LEC partially. The activated calpains can induce degradation of crystallins and apoptosis of LEC, which may play a role in the mechanism of oxidative cataract. Caspase 3-dependent pathway may contribute to apoptosis of LEC induced by calpains.

Heparin drug delivery system (HEP DDS) was developed and applied to the experimental animals for the prevention of after cataract.

Thirty New Zealand rabbits (30 eyes) received phacoemulsification, and were randomly divided into 3 groups: normal saline eye dripping (Group A); posterior chamber implantation of carrier drug delivery system (Group B) or HEP DDS (Group C). All eyes were examined by slit-lamp microscopy, the blood and aqueous humor heparin concentrations were measured and the wet weight of posterior capsules was weighed.

(1) In the 12th week after the operation, the number of eyes developed posterior capsule opacification in Groups A, B, and C was 10, 10 and 4, respectively; with statistically very significant difference (χ²=14.09, P=0.001). (2) Twelve weeks after operation, the average wet weight of the posterior capsules in these 3 groups was (158±5) mg, (160±9) mg and (20±3) mg, respectively; with statistically very significant difference (F=1168.99, P=0.000). (3) The average value of the aqueous humor heparin concentration in group C was (26±12) mg/L. (4) Light and electron microscopy did not revealed any active cell proliferation in the posterior capsules in Group C; the conjunctiva, cornea, iris, ciliary body and retina remained intact, without obviously toxic reaction. (5) No intraocular hemorrhage occurred during the follow-up period.

The implantation of HEP DDS into the posterior chamber can raise the aqueous humor heparin concentration to a high level and remained for a long period. This procedure can effectively inhibit the occurrence of after cataract, has few toxic and side effects, and is proved to be a safe and effective procedure.

To investigate the expression and significance of transforming growth factor-β₁ (TGF-β₁), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in human Lens epithelial cells (LEC) of diabetic cataract.

Immunohistochemical staining was used to detect the expression of TGF-β₁, MMP-2 and TIMP-2 in the diabetic LEC (23 cases) and the normal LEC (7 cases).

There was significant difference of expression of MMP-2 and TGF-β₁ between diabetic LEC and normal LEC (P<0.01). But there was no statistic significance for TIMP-2 expression (P >0.05). There was correlation between the expression of TGF-β₁ and MMP-2.

The imbalance induced by TGF-β₁ between the expression of MMP-2 and TIMP-2 may play a critical role in the pathological fibrosis of anterior and posterior subcapsula during the development of diabetic cataract.

To investigate the natural course and the final visual acuity in patients of age-related macular degeneration with choroidal neovascularization.

Thirty five eyes of 29 patients diagnosed as AMD with occult choroidal neovascularization were studied. The eyes which had laser, radiation therapy or surgical treatment were excluded. Visual acuity, fundus examination, fluorescent angiography (FFA) and perimetry test were performed at the first time visit and 5 years following-up at an interval of 1 to 3 months. The average following-up period of time was 8 years (range from 5 to 16 years) which started in 1985 and ended in 2001.

Initial visual acuity 0.1 or less was shown in 10 eyes (28.5%, only hands movement can be seen in 4 of 10 eyes), 23 eyes(65.7%) with visual acuity of 0.1 or less (14 eyes with vision acuity of hands movement) on the last visit. 7 of 35 eyes, the final visual acuity improved more than 2 lines, 12 eyes remained the same and 16 eyes had been lost vision more than two lines. At the baseline examination, 25 eyes had macular hemorrhages and 4 eyes had vitreous hemorrhages. The fundus hemorrhages occurred repeatedly 1 to 4 times in 23 eyes during following-up, the macular hemorrhages was detected in 7 eyes at the last visit. At the initial fundus examination, fibrotic membranes were found in 7 eyes; all 35 eyes had disc form scarring with various size in most recent following-up.

The natural course of Age-related macular degeneration with choroidal neovascularization is prolonged and the final visual prognosis was poor especially in the cases with recurrent macular hemorrhages.

To investigate the surgical timing and effects for vitreous hemorrhage caused by blunt ocular trauma.

116 eyes of vitreous hemorrhage caused by ocular blunt trauma were divided into two groups, non-vitrectomy group (46 eyes) and vitrectomy group (70 eyes). The treating results and follow-up results were compared.

50 of 70 eyes (71.4%) in vitrectomy group and 10 of 46 eyes(21.7%) in non-vitrectomy group achieved a visual acuity of 0.1 or better over a follow-up period of 1 month, which indicated a significant difference between two groups. In the non-vitrectomy group, visual acuity of 10 eyes were better than 0.1, 7 eyes (70.0%) of them improved within 2 weeks of injury. In surgery group, 27.1% underwent only vitrectomy and 70.9% needed combining with other manipulations. In vitrectomy group the retina detachment was found in 3 eyes (4.3%) post-operatively and were cured by second operation. In non-vitrectomy group, the retinal detachment was found in 14 eyes (30.4%) during the follow-up period, and 8 eyes (72.7%) of 11 eyes received operation were cured.

Early vitreous surgery can improve curative effect for vitreous hemorrhage caused by blunt ocular trauma when no improvement was observed after applied drugs 2-3 weeks.

To evaluate the current situation of corneal donors and possible way to improve the corneal donation program in the city of Chongqing.

(1) The 1400 residents of Chongqing were randomly surveyed to understand their ideas and attitudes towards corneal donation and to evaluate the correlation between their knowledge and their participation in the program in May ~ December, 2001. (2) The sociology data of 1278 registered donors in the Chongqing Eye Bank were analyzed from March 1999 to December 2001. (3) The social-medical data of 37 eye donors were analyzed from March 1999 to December 2001.

(1) Results of the random survey: the response rate was 79.14% (1108/1400), 1072 (76.57%) questionnare met the criteria for further analysis. 47.20% of 1072 persons would like to be eye donors, 62.69% supported eye donation, 32.55% would only donate their cornea, but 90.49% did not register to be a donor at present. Of those surveyed, about 2/3 were not familiar with the Chongqing Eye Bank, most did not know how to become an eye donor, and 75.28% supported local legislation to regulate donation. (2) Data of 1278 registered eye donors: 550 were males and 728 were females, 61.89% were 30 years old or younger, most were students, teachers, medical staff or white-collared workers in general, and more than half had a university level education. (3) Data of 37 eye donors: 25 were males and 12 were females, age range was 20 ~ 88 years old, the main causes of death were malignant tumors and cardio-cerebral vessel disorders, 59% had nine or less years of education while only 11% had a university level education, and nine were also cadaver donors. The clinical utilization rate of eyes was 73%.

(1) There are a significant number of people who are willing to be eye donors. Education level and age do not affect their decision to become donors. (2) Only a small percentage of the mass are registered eye donors. (3) Legislation regarding eye donation should be enacted as soon as possible. (4) Corneal donations could be higher than those at present.

To observe the therapeutic effect of rAd-gfp-bcl-X_L on retinal degeneration (RD) in mice.

rAd-gfp-bcl-X_L granules were injected into the subretinal space of right eyes of RD mice in Group A (postnatal days 10, p10) and Group B (postnatal days 22, p22) (n=20). The morphological changes were observed by light microscopy and transmission electron microscopy at 15-30 days post-injection. Frozen section was used to observe the expression of GFP. The level of bcl-X_L mRNA and bcl-X_L protein were assayed by RT-PCR and immunohistochemistry assay.

In frozen section, GFP was observed in the treated eyes under fluorescence microscopy, indicating that rAd-gfp-bcl-X_L had transfected into the retinal cells successfully and that bcl-X_L was expressed. In Group A, an increased expression of bcl-X_L mRNA and Bcl-X_L protein were observed in the treated eyes. The outer nuclear layer of the treated eyes in Group A was thicker than that of the control eyes at 30 days post-injection. The inner segment of treated eyes in Group A was healthier than that of control eyes, as indicated by transmission electron microscopy. No difference was found in the thickness of retina between treated eyes and control eyes in Group B.

The bcl-X_L can be expressed and have an anti-apoptosis effect when the rAd-gfp-bcl-X_L particles are injected into the subretinal space of RD mice's eyes at an early stage. But the expression and protective effects were not observed in RD mice at a later stage. The therapy of rAd-gfp-bcl-X_L for retinal degenerative diseases still needs further study.

To study the phenotypic changes of lymphocytes and their subsets in patients with severe acute respiratory syndrome (SARS), in order to provide new monitoring indicators for the diagnosis and treatment of SARS.

The expression of peripheral blood lymphocytes and their subsets in 38 patients with SARS was measured by multicolor flow cytometry combined with blood analyzer.

Absolute lymphocyte count decreased in 84% of patients. The absolute counts of all kinds of lymphocyte subsets [T/B lymphocytes, natural killer (NK) cells] all decreased to varying degrees, among which T cells decreased the most (accounting for 95%); In T cells, T helper/inducer cells (T₄) decreased (100%) more than T suppressor/NK cells (T₈) reducers (87%). From the distribution of various lymphocytes, the percentage of T lymphocytes decreased the most (58%); The percentages of B cells and NK cells were mainly relatively normal or increased, and the percentages of B cells and NK cells were relatively decreased, accounting for 2.6% and 5.3% respectively; In T cells, T₄The percentage of cells decreased (82%) was higher than T₈The cells were reduced (34%), and the ratio of the two was reduced in 44%.

The total number of lymphocytes and their subpopulations in SARS patients were destroyed, mainly T lymphocytes, and T₄Cells compared to T₈Cells are severely damaged; The absolute counts of lymphocyte subsets changed more significantly than the relative counts.

Chest X-ray findings of severe acute respiratory syndrome (SARS) are reported.

The chest X-ray findings and characteristics of 105 patients with SARS were analyzed. All patients were diagnosed and treated between January 30 and March 31, 2003, and met the trial criteria for clinical diagnosis of SARS established by the Ministry of Health of the People's Republic of China, and had complete chest X-ray data during hospitalization. The lesions were classified into 4 types according to their early manifestations and progression. (1) Localized type: from a single local lesion in the lung to the development or widespread distribution of the lesion. (2) Multiple type: multiple flaky or (and) nodular lesions in the lungs are seen in the early stage. (3) Interstitial-parenchymal type: interstitial inflammation of the lung is the main manifestation in the early stage, followed by obvious exudative lesions of the pulmonary parenchyma. (4) Interstitial type: pulmonary interstitial abnormalities are the main manifestation.

The chest X-ray manifestations of SARS patients were varied. 75 cases showed exudative consolidation lesions in the lung, including 57 cases of localized type and 18 cases of multiple type. Twenty-five cases showed signs of interstitial inflammation followed by significant intrapulmonary consolidation. Five cases mainly showed interstitial inflammation of the lung. The time from the first intrapulmonary lesion to the complete absorption of the lesion was 7 to 46 days, with an average of 19 days.

The X-ray manifestations of SARS were mainly exudative lesions of lung parenchyma and interstitial inflammation. The lesions developed rapidly in the early stage, and the peak of intrapulmonary consolidation reached 7~10 days after the first diagnosis, and after that most patients could completely absorb them. The vast majority of patients have a good prognosis, and no sequelae have been seen at present.

To investigate the early X-ray and CT findings of severe acute respiratory syndrome (SARS).

The early X-ray and CT findings of 28 patients with SARS were analyzed. The patients underwent X-ray and CT examination within 1 to 3 days of onset. CT examination consists of conventional spiral scan and high-resolution CT (HRCT).

There were abnormal findings in chest CT examination in 28 cases. Chest X-ray showed lesions in 60.7% (17/28). The imaging findings can be divided into: (1) Single small patch lesions: 82.1% (23/28). On CT images, the lesions were divided into three types: ① round ground glass-like density lesions in 20 cases; ② Two cases of ground glass density lesions in the form of pulmonary lobules; ③ One case of small patch consolidation imaging. (2) Two cases of multiple small patch-like ground glass lesions. (3) Large patch images in 3 cases. A total of 31 lesions were found in 28 patients, 7 of which were distributed in the upper lobe, accounting for 22.6%; 3 in the middle lobe, accounting for 9.7%; There are 21 lower lobes, accounting for 67.7%. The maximum diameter of the lesions was 2.0~3.5cm.

The early X-ray and CT manifestations of SARS are mainly single small patch images in the lung. CT showed that the morphology of the lesions was like round ground glass density lesions.

To study the characteristics of T lymphocyte changes in patients with severe acute respiratory syndrome (SARS), explore its pathogenesis, and improve the early diagnosis of SARS.

The anticoagulant blood of 30 patients diagnosed with SARS and 56 healthy blood donors were collected and labeled with specific fluorescent antibodies. The CD was detected by flow cytometry⁺₄And CD⁺₈The changes of T lymphocyte subsets were observed in 2 patients with SARS after recovery⁺₄And CD⁺₈of T lymphocyte subsets and compared with T lymphocyte subset alterations in patients infected with human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV).

CD in SARS patients compared with normal people⁺₄T lymphocytes ("T₄cells ") and CD⁺₈T lymphocytes ("T₈Cells ") were significantly reduced [(284±187) cells/mm³and (288±167) /mm³], T of all SARS patients₄Cell counts were lower than normal (the lowest was only 49 cells/mm³), the T of 2 SARS patients during the recovery period₄and T₈Cells return to normal; Compared with those infected with HIV, CMV, and EBV, the T of SARS patients₄Cells and T₈The number of cells was also significantly reduced, while T₈The decrease in cell numbers is more severe; Compared with normal people, HIV-infected people T₄The number of cells was also significantly reduced, but the T of HIV, CMV, and EBV infected people₈The number of cells increased significantly.

The number of T lymphocytes in SARS patients was significantly reduced, but reversible; T₄and T₈The detection of cells is helpful for early diagnosis of SARS patients.

To explore the pathogen of SARS outbreak in Guangdong and other places at the end of 2002.

The autopsy specimens of 7 dead SARS patients from Guangdong, Shanxi, Sichuan and Beijing were observed by ultrathin section electron microscopy. The lung tissue extracts of patients were inoculated into 293 cells (human embryonic kidney transformed cell line), and the pathogens were isolated. The pathogens in the tissues of patients and the isolated pathogens were immunologically identified.

Chlamydia-like inclusion bodies, reticulosomes, intermediates and protosomes were observed in lung (7 cases), spleen (2 cases), liver (2 cases), kidney (3 cases) and lymph node (1 case) of deceased patients. Coronavirus-like particles and unknown matrix-like structures were also observed in lung tissues of 3 patients. Coronavirus RNA polymerase gene fragments were amplified from lung tissues of two patients by RT-PCR. The lung tissue extract was inoculated into 293 cells for culture, and the second generation cells were taken for electron microscopy observation. The presence of chlamydia-like particles and inclusion bodies was also found. Chlamydia-like particles found in tissues and cultured cells of patients could not react with specific antibodies of Chlamydia genus and antibodies against Chlamydia pneumoniae and Chlamydia psittaci, suggesting that they may be new chlamydia-like factors.

Chlamydia-like and coronavirus-like particles found in tissues and cultured cells of dead patients may be the main pathogens of SARS outbreaks in Guangdong and other places.

A fluorescent polymerase chain reaction (F-PCR) method for the detection of severe acute respiratory syndrome (SARS) coronavirus was established and its clinical application value was discussed.

According to the published SARS coronavirus gene sequence, we designed and synthesized primers and probes by ourselves, and applied the developed SARS F-PCR diagnostic kit to detect 115 clinical mouthwash samples.

The PCR amplified product was sequenced and proved to be identical to the SARS coronavirus gene sequence. Among 67 confirmed SARS patients, 49 mouthwash were positive; Out of 18 close contacts, 8 mouthwash were positive; All 30 healthy subjects were negative for mouthwash.

The established F-PCR method for detection of SARS coronavirus can be a rapid, accurate and effective detection method for SARS screening and early diagnosis.