Expression And Application Of Ciliary Neurotrophic Factor(CNTF) In Silk Glands Of Silkworm

The optic nerve disease leads to poor vision characterized by retinal ganglion cells（RGCs）injury,even blindness.There are different kinds of the diseases like diabetic retinopathy,age-related macular degeneration and glaucoma.The current treatment methods mainly include adding eye drops and surgery,however,they all have certain limitations,such as poor compliance and unsatisfactory surgical effect.Therefore,the development of more efficient treatment has become an important topic in the field of retinal ganglion cells injury research.It is difficult to regenerate axons directly after retinal ganglion cells injury,which can lead to disruption of optic nerve cells transmission.Moreover,it can lead to blurred or even loss of vision.Fortunately,the researches have shown that the ciliary neurotrophic factor（CNTF）can protect RGCs and promote the neurite regeneration effectively,therefore,CNTF has a good therapeutic effect.Although CNTF exists widely in vivo,its content is very low.Obtaining large amounts of active CNTF through exogenous expression is the basis of therapeutic application.Silkworm is an ideal expression system for exogenous proteins.Its silk gland is an organ for efficient synthesis of silk proteins,often used as a bioreactor to produce a variety of exogenous proteins.Moreover,silk has the advantages of good biocompatibility and excellent mechanical properties,and has been widely used in tissue engineering,medicine and drugs.In this study,the silkworm silk gland bioreactor was used to efficiently produce human ciliary neurotrophic factor（CNTF）,and biological materials containing CNTF were prepared from silkworm cocoons and used for the culture of retinal ganglion cells in vitro and so on.The main research results are as follows:1.Construction and characterization of transgenic silkworm CNTF strainCNTF has a wide range of biological functions,including protecting neuronal survival,promoting neuronal cell proliferation and inducing axon regeneration.In this study,the human CNTF gene sequence was downloaded from the NCBI and synthesized artificially after the codon optimization of silkworm.The transgenic vectors specifically expressing CNTF in the middle silk glands（MSG）and posterior silk glands（PSG）of silkworm were constructed,and the plasmid was transferred into the genome of silkworm embryo by microinjection technology.Two transgenic positive individuals were obtained by fluorescence screening named as CNTF（MSG）and CNTF（PSG）.The results of Western Blot analysis showed that CNTF was highly expressed in transgenic silkworm cocoons.Secondly,the genome of transgenic moth was extracted for PCR.The results showed that CNTF gene was successfully inserted into silkworm genome.Thirdly,the specific expression of CNTF in the middle and posterior silk glands of silkworm was detected by RT-q PCR.Then,the CNTF of CNTF（MSG）silkworm was localized in sericin layer,and the CNTF of CNTF（PSG）silkworm was localized in fibroin layer.Two kinds of transgenic silkworm were hybridized to increase the content of CNTF in cocoon,and the transgenic silkworm expressing CNTF in MSG and PSG was obtained.The content of CNTF in each gram of cocoon after hybridization was estimated to be 3.2 mg by SDS-PAGE staining.Finally,economic character investigation and SEM observation of silkworm silk between transgenic CNTF and wild-type（WT）showed that there were no significant difference,indicating that the expression of exogenous CNTF gene had no significant effect on the formation of silk fibers.2.Bioactivity assays of CNTF expressed by transgenic silkwormIn order to detect the activity of CNTF expressed by silkworm,the silk protein solution was extracted and the cell activity was analyzed.The results of Live-Dead assay showed that CNTF silk extract had no toxic effect on mouse neural stem cells（NE-4C）and mouse retinal ganglion cells（RGC-5）.Then the results of CCK-8 and Ed U reagent staining showed that CNTF could effectively promote the proliferation of NE-4C and RGC-5 cells.Next the cell scratch test showed that CNTF had promoted migration activity on NE-4C and RGC-5.Finally,detecting the expression level of GAP43 by Western Blot analysis and observing Actin-Tracker Red-555 fluorescence staining,the results showed that CNTF had great activity to promote the growth of nerve axons in RGC-5 cells.3.Construction of CNTF sericin hydrogel and its application in nerve cellsSericin hydrogel was prepared from CNTF silk protein solution by low temperature dialysis.Western Blot results showed that CNTF existed in CNTF sericin hydrogel.The hydrogel was freeze-dried and observed by SEM.The cross section of the freeze-dried gel showed a porous network structure,the pore size was about 31.43μm,and the longitudinal section was gully.The hydrogel porosity was measured at 33.3%,and swelling behavior analysis showed that it could swell to 12.8 times of the original,indicating that it had good hydrophilicity.The number of cells shed was detected by CCK-8,and the results showed that the number of cells shed on the hydrogel was less,indicating that the hydrogel material could promote cell adhesion.The cells on the hydrogel were cultured in a low-concentration serum medium,the results of CCK-8 and DAPI staining showed that CNTF sericin hydrogel could promote the proliferation of RGC-5 cells.Cell damage model was prepared by treating cells with H₂O₂ reagent,flow cytometry was used to analyze cell apoptosis,and the results showed that the proportion of viable cells treated with H₂O₂ in blank cell culture hole was the lowest,only 17.22%,and the proportion of living cells in WT hydrogel treatment group was 39.63%,but the survival rate of cells treated with CNTF hydrogel was higher（46.37%）.This explains that CNTF sericin hydrogel could protect the survival of the injured RGC-5 cells.In conclusion,transgenic silkworm strains expressing human CNTF protein in middle and posterior silk glands were successfully obtained using silkworm silk gland bioreactor in this study.It was verified that CNTF protein in transgenic silkworm cocoon has biological activities of promoting cell proliferation,migration and nerve axon growth.CNTF sericin hydrogel was prepared,which could promote the adhesion,proliferation and survival of RGC-5 cells after injury.This study provides a new method for the efficient production of bioactive CNTF proteins,and the combination with silk materials will help to advance the preparation of silk drug delivery system for optic nerve diseases and other aspects of research.