OBJECTIVE: To investigate the effects of basic fibroblast growth factor (bFGF) and its receptor antibody on the ultrastructure of human lens epithelial cells (HLECs). METHODS: Transmission electron microscopy was used to observe the ultrastructural changes of normal HLECs and bFGF after the action of bFGF and the receptor antibody blocking the action of bFGF in HLECs. Results: Normal HLECs had more mitochondria, rough endoplasmic reticulum, free ribosomes and a few microfilament structures, intact nuclear membrane, and uniform chromatin. bFGF action showed intact cell membrane, more local protrusions, significantly more mitochondria and free ribosomes, denser matrix, more microfilament structures near the nucleus, intact nuclear membrane, uniform chromatin, and enlarged nucleolus. After blocking the effect, it showed that the cell protrusions disappeared, the cell membrane was obviously destroyed, a large number of mitochondria were cavitated, the cristae disappeared, the matrix was reduced and degenerated in a cloudy manner, the endoplasmic reticulum was obviously reduced to disappeared, the nuclear membrane was obviously destroyed, and some cells had unclear structures and tended to die. Conclusion: The pro-proliferative effect of bFGF on HLECs can be blocked by FGF receptor antibodies. The occurrence of posterior capsule clouding is related to postoperative lens epithelial cell proliferation, migration and fibrosis, and the posterior capsule changes in the lens are closely related to the role of growth factors, especially the role of basic fibroblast growth factor (bFGF) receptors, which cannot be ignored. Blocking the FGF receptor can inhibit the proliferative effect of bFGF on HLECs [1], and we investigated the changes in cellular ultrastructure caused by its action. 1, Materials and methods 1.1, Fetal lens epithelial cell culture Fresh 2 eyeballs from the same fetus were rinsed well with saline, then rinsed with gentamicin, placed in a clean glassware, cleared of periocular tissue, and the conjunctiva was cut. The treated eyeballs were rinsed well with diluted gentamicin solution, placed in another clean glass culture vessel, and the sclera was cut circumferentially on an ultra-clean bench at a distance of approximately 4 cm from the corneal limbus, and the upper part (the cornea was used to culture corneal epithelial cells) was removed and the lens was removed. The anterior capsule was separated along the equatorial part of the lens and placed in a 27 mm diameter plastic culture dish with the cell surface facing the bottom, and a few drops of 150 g/L DMEM culture solution (pH 7.2) was added. The capsule was cut into pieces as much as possible with iris scissors, spread and dispersed, and then placed in a CO2 chamber (MCO17AI, SANTO, Japan) for about 4 h. Then, the culture solution was gently replenished with about 2.5 mL of culture solution. Continue to culture (culture conditions were 37°C, 50mL/L CO2, saturation temperature. Cell growth was observed daily under the microscope, and the culture medium was changed for 7-10 d. Transfusion culture: when the cell growth is fused, aspirate the culture fluid, rinse with Hanks solution twice, add 2.5g/L trypsin solution 1.5mL, digest 4~8min aspirate, add the culture fluid containing 150g/L FCF DMEM repeatedly blow to make the cells suspended, publish evenly, then divide to several culture dishes to continue the culture. 1.2, Preparation of cell specimens Experiments were performed using 2 generations of cells. The coverslips cut into small pieces were sterilized and placed in a 27mm diameter glass culture vessel. Fetal lens epithelial cells, cells with bFGF (100μg/L) added and cell suspension with bFGF (100μg/L) and bFGF receptor antibody (5mg/L) added were dropped onto the coverslip pieces in the culture vessel to make 3 different samples, which were placed in a CO2 incubator for 4d. 1.3. Transmission electron microscopy specimen production When the coverslips were full of cells, the culture medium was discarded and rinsed twice with Hanks’ solution. The cells grown against the wall were scraped off with a rubber spatula, the supernatant was removed after centrifugation, fixed with 30g/L haptogaldehydes, fixed with 10g/L osmium acid, dehydrated by ethanol step by step, embedded by permeation with Epon812, polymerized for 72h in a 60℃ incubator, sectioned by LKBB400 ultrathin sectioning machine, double-stained with uranyl acetate and lead citrate, observed and photographed under philips EM 400T transmission electron microscope. 2, Results 2.1, Fetal lens epithelial cells Fetal lens epithelial cells (2nd generation) showed normal intracellular mitochondria, rough endoplasmic reticulum, free ribosomes and a few microfilament structures; the nuclear membrane was intact, chromatin was uniform, and the number of organelles was limited, suggesting that HLECs are still functional, but they have poor proliferation ability. 2.2. Fetal lens epithelial cells with bFGF (100μg/L) The ultrastructure of fetal lens epithelial cells cultured with bFGF was as follows: the cell membrane was intact, with more local protrusions in the form of short rods or fingers; the intracellular organelles were abundant, especially the mitochondria with good function were obviously increased, and the matrix was dense, and the free ribosomes were increased, and there were more microfilaments along the long axis of the cell near the nucleus. There were more microfilaments arranged in parallel along the long axis of the cell near the nucleus; the nuclear membrane was intact, the chromatin was coarse and uniform, and the nucleolus was enlarged. The increase in organelles indicated that the addition of bFGF exerted a pro-proliferative effect on the cells, resulting in a strong metabolic function and enhanced mitotic ability; at the same time, it also indicated that bFGF had a certain differentiation effect on the cells, such as the microfilament structure along the long axis, which is a typical feature after cell differentiation. 2.3. When bFGF (100μg/L) and FGF receptor antibody (5mg/L) were added to fetal lens epithelial cells, the cell ultrastructure showed that the cell protrusions disappeared, the membrane was obviously destroyed, a large number of mitochondria were cavitated, the cristae disappeared, the matrix was reduced and degenerated in a cloudy manner, the endoplasmic reticulum was also obviously reduced or even disappeared, only a few microfilaments were present and were broken The cellular organelles were obviously reduced and destroyed, and some of them disappeared; the nuclear membrane was obviously destroyed; some cells had unclear structure and even tended to die, suggesting that the bFGF receptors were antagonized by the antibodies in the antibody neutralization experiment, so that the effect of bFGF disappeared and the cell metabolism basically disappeared and tended to die. 3, Discussion Lens epithelial cells are the only kind of cells in the lens that contain more abundant organelles, and their environment is also different from other epithelial cells, which play an important role in maintaining lens metabolism. While the epithelial cells in the center of the anterior capsule are relatively quiescent, the epithelial cells at the equator proliferate and extend toward the anterior and posterior poles of the lens, gradually differentiating into lens fiber cells and forming the multilayered structure of the lens, with changes such as elongation of the cell body and reduction of organelles during the process. The literature reports that electron microscopic observation of the lens reveals that the anterior capsule and equatorial part of the capsule are tightly connected to the lens cells, while the posterior capsule is thinner and has no cellular structure. The presence of a large number of microvilli in the lens epithelium expands the surface area of the cells, providing a venue for the exchange of material between the cells and the atrial fluid, thus accelerating the process of material exchange between the lens fibers and the atrial fluid. Mitochondria are rod-shaped or ovoid-shaped in cells in good functional state, and are more numerous, and can become granular or agglomerate into larger clusters when they are in poor function or metabolism; microtubules and microfilaments form the cytoplasmic skeleton, and they have an impact on cell membrane movements such as swallowing and immune responses, in addition to supporting cell morphology and being related to cell motility. In general, elongated cells that have become fibrotic have a different ability to respond to bFGF, and cell elongation is also shown to be consistent with early fiber differentiation activity. When cataract occurs under the posterior capsule, the peripheral lens epithelium migrates posteriorly to form multiple layers of patchy, swollen cells and some elongated cells, when abundant capsule-like matrix accumulates beneath the capsule and between the cells, as well as important changes in the rough endoplasmic reticulum, Golgi complex, microfilaments, and apparent capsule folds and apoptotic cells; this has some connection with similar changes in posterior capsule clouding after cataract surgery . And recent studies of anterior capsular clouding have yielded similar results. In this experiment, we observed by transmission electron microscopy that the organelles increased after the addition of bFGF, indicating that it exerted a pro-proliferative effect on the cells, resulting in a strong metabolic function and enhanced mitotic capacity, and that bFGF also had a certain differentiation effect on the cells, such as the ultrastructure of microfilaments arranged along the long axis, which is a typical feature of the differentiated cells. Under the same conditions, when bFGF was added together with antibodies to FGF receptor, the organelles were obviously reduced and destroyed, some disappeared, the nuclear membrane was obviously destroyed, and some cells had unclear structure and even tended to die, suggesting that the bFGF receptor was antagonized by the antibodies in the antibody neutralization experiment, so that the effect of bFGF disappeared, cell metabolism basically disappeared and tended to die. The fibroblast growth factor receptor consists of an extracellular ligand region, a helical transmembrane region, and an intracellular region, and the four FGF receptor genes can produce 48 receptor isoforms. fGFR1 expression decreases with age in lens epithelial cells, but enhanced expression of FGFR1 after FGF action plays an important role in cell development and fibrosis. Blocking the FGF receptor, in any way, inhibits the proliferation of lens cells. Electron microscopy revealed experimental evidence that bFGF plays an important role in maintaining capsule membrane support for lens epithelial cells. bFGF’s pro-proliferative effect on lens cells is mediated by its binding to the FGF receptor through the MAPK/ERK signaling system. bFGF and its receptor play an important role in lens epithelial cell proliferation and differentiation. Our results confirm the pro-proliferative effect of bFGF on HLECs and its ability to be blocked by antibodies to its receptor, and indirectly suggest that bFGF exerts its biological effects through binding to the receptor on the cell surface. This provides a basis for in vivo experiments to elucidate the role of bFGF and the “receptor binding” theory at the molecular level.