KR102114787B1 - Preparing method for 3 dimension corneal endothelial graft comprising ribonuclease 5 overexepressing corneal endothelial cells - Google Patents

Abstract

The present invention relates to a method for producing a three-dimensional corneal endothelial implant containing ribonuclease 5 highly expressed corneal endothelial cells, and more specifically, corneal endothelial cells highly expressed with ribonuclease 5 (RNase 5) In the case of a 3D bio-printed corneal endothelial implant, zonular occludens-1 (ZO-1) expression was increased compared to corneal endothelial cells in which ribonuclease 5 (RNase 5) was highly expressed, resulting in improved cell phenotype. As it is confirmed that Na ⁺ -K ⁺ ATPase expression is also increased, it is possible to effectively prevent and treat swelling of the damaged cornea, and thus, 3D corneal endothelial transplantation using corneal endothelial cells highly expressed with ribonuclease 5 (RNase 5) The sieve can be used for effective damage to the cornea.

Description

Preparing method for 3 dimension corneal endothelial graft comprising ribonuclease 5 overexepressing corneal endothelial cells}

The present invention relates to a 3D bioprinted corneal endothelial implant using corneal endothelial cells highly expressed with ribonuclease 5.

Anatomically, the corneal endothelial tissue forms a single-layered corneal endothelial layer by forming hexagonal corneal endothelial cells with tight junctions between cells, and the intercellular closet blocks water inflow to the cornea and corneal edema, Since the Na-K ATPase pump is expressed on the cell surface to pump out moisture that has flowed into the cornea, it also prevents edema of the cornea and maintains transparency. Therefore, for the intrinsic function of the corneal endothelium, the number of cells must be sufficient, the cell-to-cell adhesion must be well developed, and the Na-K ATPase expression on the cell surface must be distinct.

However, regeneration of damaged corneal endothelial cells in vivo is extremely limited, and when endothelial cells are damaged by various diseases such as Fuchs' corneal dystrophy, trauma, and phacoepithelial keratosis, corneal swelling and turbidity can cause serious vision. It can also cause loss.

These human corneal endothelial cells are stationary in the G1 phase of the cell cycle throughout their lives, and at the same time, the expression of several regulators, such as the p53 gene, which inhibits cell cycle progression, has increased, so most cells undergo cell proliferation and cell migration during the damage healing process. On the contrary, as the corneal endothelial cells repair the damaged area through an increase in cell migration and cell size, the cornea swells several times the thickness of the normal cornea and becomes opaque and loses its function.

Recently, it is known that the stem cells of the corneal endothelial cells exist between the corneal endothelial and the fibrous line, and cells stained with the stem cell marker nestin at this transition zone have been discovered, and the corneal endothelial cells are also found in the human body. Although it can be suggested that it can be proliferated in, there is no method to proliferate corneal endothelial cells in vivo, so there is no alternative to corneal endothelial cell damage to treat corneal endothelial cell damage. .

Republic of Korea Patent No. 10-2014-0031814 (2014.03.13. Public)

As described above, the damage of the corneal endothelial cells is directly related to the prognosis of vision, but the corneal endothelial cells have many difficulties in treating corneal endothelial damage because they do not have their own proliferative ability, unlike other cells. The present invention relates to a method of manufacturing a 3D bioprinted corneal endothelial implant using corneal endothelial cells highly expressed with ribonuclease 5 for transplantation.

The present invention is a step of overexpressing ribonuclease 5 (Ribonuclease 5) on corneal endothelial cells (first step); Dispersing corneal endothelial cells overexpressed in the first step of ribonuclease 5 in bioink (second step); Separating the bioink in which the corneal endothelial cells of the second step are dispersed from livestock and bioprinting the lyophilized amniotic membrane (third step); And it provides a three-dimensional bio-printed corneal endothelial implant manufacturing method comprising the step of cross-linking by irradiating ultraviolet rays to the bio-printed amniotic membrane of the third step (step 4).

In addition, the present invention provides a corneal endothelial implant in which a corneal endothelial cell overexpressing ribonuclease 5 according to the method for preparing the corneal endothelial implant is 3D bioprinted on the amniotic membrane.

According to the present invention, in the case of corneal endothelial transplantation in which a corneal endothelial cell having high expression of ribonuclease 5 (RNase 5) is 3D bioprinted, than corneal endothelial cell having high expression of ribonuclease 5 (RNase 5) As zonular occludens-1 (ZO-1) expression was increased, cell phenotype was improved and Na ⁺ -K ⁺ ATPase expression on the cell surface was also confirmed to be increased, so it is possible to effectively prevent and treat edema of the damaged cornea. A three-dimensional corneal endothelial implant using RNase 5 highly expressed corneal endothelial cells can be used for effective damage to the cornea.

s.e.m.].
도 6은 배양된 사람 각막 내피세포의 증식에 있어 리보핵산분해효소 5(RNase 5) 과발현 효과를 확인한 결과로, 도 6A는 mock과 비교하여 RNase 5 발현 벡터(R5-HCECs)로 형질전환된 HCECs의 형질전환-페세지 1에서 상대적인 BrdU 흡수량을 확인한 결과이며, 도 6B는 대조군 HCECs와 mock 그룹과 비교하여 R5-HCECs의 성장 곡선은 배양 배지에서 5일 이상 유지되는 것을 확인한 결과로, RNase 5의 과발현은 BrdU 흡수를 유의적으로 증가시켰으며(A), R5-HCECs (0.5 ㎍ 벡터)의 성장속도를 가속화시켰음을 확인한 결과이다 [(A) **p < 0.01. n = 4 independent experiments. (B) **p < 0.01 and *p < 0.05, vs. control HCEC. ^## p < 0.01 and ^# p < 0.05, vs. mock. n = 5 independent experiments.].
도 7은 리보핵산분해효소 5(RNase 5) 발현 벡터로 형질전환되어 배양된 인간 각막 내피세포(HCECs)에서 마이크로 RNA (miR)-21, miR-23a 및 miR-27a의 발현 수준을 확인한 결과로, 도 7A 내지 도 7C는 miR-21의 발현 변화수준을 확인한 결과이며, 도 7D 내지 도 7F는 miR-23a의 발현 변화수준을 확인한 결과이며, 도 7G 내지 도 7I는 miR-27a의 발현 수준을 확인한 결과로, 상기 발현수준은 RNase 5 발현 벡터(0.2 ㎍ 및 0.5 ㎍, R5-HCECs)로 24시간 및 48시간 동안 형질전환된 HCECs(transfection-passage 0; t-P0)와 72시간 동안 배양 배지에서 유지시킨 R5-HCECs(passage; t-P1)에서 miRs의 변화를 실시간 RT-PCR을 수행하여 정량한 결과이다. 중요한 것은 R5-HCECs 내 miRs의 상향 조절은 형질전환(t-P0)시기 보다 페세지(t-P1) 후 더욱 두드러지게 나타났다[**p < 0.01 and *p < 0.05 (ANOVA followed by Bonferroni's post-hoc analysis). n = 3 independent experiments. Values represent the mean

s.e.m.].
도 8은 RNase 5 발현 벡터로 형질전환된 배양된 인간 각막 내피세포 (HCECs)에서 리보핵산분해효소 5(RNase 5)의 표적 유전자 후보를 확인한 정량적 실시간 RT-PCR 분석 결과로, 도 8A는 PDCD4 (programmed cell death protein 4; A)의 발현 수준을 확인한 결과이며, 도 8B는 PTEN(phosphatase and tensin homolog)의 발현 수준을 확인한 결과이며, 도 8C는 Cdc25A의 발현 수준을 확인한 결과이며, 도 8D는 Cdk2AP1의 발현 수준을 확인한 결과이며, 도 8E는 Spry1(sprouty RTK signaling antagonist 1)의 발현 수준을 확인한 결과이며, 도 8F는 Spry2의 발현 수준을 확인한 결과이며, 도 8G는 Apaf1(apoptotic protease activating factor 1)의 발현 수준을 확인한 결과이며, 상기 발현 수준은 RNase 5 발현 벡터로 형질전환되어 배양된 HCECs와 mock 그룹 및 음성 대조군(N/C) 벡터로 형질전환된 HCECs를 비교한 결과이며 [**p < 0.01 and *p < 0.05 (ANOVA followed by Bonferroni's post-hoc analysis). n = 8 independent experiments. Values represent the mean

s.e.m.], 도 8H는 배양된 mock, N/C 벡터 및 RNase 5 벡터 HCECs 군에서 PDCD4, cyclin A2, cyclin D1, cyclin D3 및 cyclin E1을 확인하기 위한 웨스턴블롯 분석 결과로, 레인 7에서 12는 t-P1의 R5-HCECs이며, β-액틴을 로딩 대조군으로 사용하였으며, 동일한 레인에서는 β-액틴 밀도에 대한 상대적인 농도 값을 각 겔 아래에 기록하였다.
도 9는 리보핵산분해효소 5(RNase 5) 발현 벡터로 형질전환되어 배양된 인간 각막 내피세포(HCEC)와 대조군 HCECs에서 기능 관련 마커의 발현 수준을 확인한 결과로, 도 9A는 20세 여성 기증자(A)로부터 배양된 초대 HCECs (passage 0; P0)를 위상차 현미경으로 확인한 결과이며, 도 9B는 RNase 5 벡터 형질전환 전 및 후에 CD166 및 CD44 발현 수준을 확인한 결과로, 상기 HCEC 세포군은 CD166 및 CD44 양성을 나타낼 뿐만 아니라, 표면 마커 역시 양성을 나타낸 RNase 5 벡터 0.5 ㎍로 48시간 동안 형질전환된 HCECs (R5-HCECs)를 t-P1(transfection-passage 1)시기에서 확인한 유세포 분석결과이다 (Scale bar: 100 ㎛). 도 9C는 대조군 HCECs (P4)와 R5-HCECs (t-P1)의 위상차 현미경 분석 결과 및 상기 세포군의 Na⁺-K⁺ ATPase 및 ZO-1(zonular occludens-1)를 확인한 면역염색 결과로, 도 9Ci 및 도 9Cii의 융합성 R5-HCECs 및 대조군 HCECs 모두 비교적 섬유아세포 표현형을 나타내지만 다각형 세포 형태와 세포 간 단단한 접합부는 대조군 HCEC 보다 R5-HCEC에서 더 많이 확인되었다. Na⁺-K⁺ ATPase 발현은 세포의 측면 막에서 발견되었으며, HCECs 보다 R5-HCECs에서 현저하게 많이 나타났다. 또한, 도 9Cx와 같이 이들 모양 중 일부는 타이트한 교차점에서 ZO-1 발현에 따라 표준형으로 확인되었다(흰 점선 경계). 이미지 iv, vi, viii 및 x는 이미지 iii, v, vii 및 ix를 각각 확대한 것이며, 세포 핵 시각화를 위해 DAPI를 사용하였다[Scale bars: 100 ㎛ (흰색), 20 ㎛ (노란색)]. 도 9D는 ATP1A1(ATPase Na⁺-K⁺ transporting subunit alpha 1) 유전자 발현을 정량적으로 확인한 실시간 RT-PCR 분석결과이며, 도 9E는 TJP1(tight junction protein 1) 유전자 발현을 정량적으로 확인한 실시간 RT-PCR 분석결과이며, 도 9F는 ALCAM(activated leukocyte cell adhesion molecule; i.e. CD166) 유전자 발현을 정량적으로 확인한 실시간 RT-PCR 분석결과이며, 도 9G는 CD44 유전자 발현을 정량적으로 확인한 실시간 RT-PCR 분석결과이다. 상기 유전자 발현량을 확인한 결과들은 R5-HCECs에서 발현된 유전자량을 mock 군 및 음성 대조군 벡터로 형질전환된 HCECs와 비교한 결과이다. [**p < 0.01 and *p < 0.05 (ANOVA followed by Bonferroni's post-hoc analysis). n = 8 independent experiments. Values represent the mean

s.e.m.]. 도 9H는 mock, N/C 벡터 및 RNase 5 벡터로 형질전환된 HCECs 세포에서 Na⁺-K⁺ ATPase, ZO-1, C166 및 CD44 발현을 확인한 웨스턴블롯 분석결과로, β-액틴을 로딩 대조군으로 사용하였으며, 동일한 레인에서 β-액틴 밀도에 대한 상대 농도 값을 각 겔 아래에 기록하였다.
도 10은 무 세포 양막(AM) 및 이식 가능한 3차원(3D) 바이오 프린트된 각막 내피 이식체의 선명도 및 조직학적 분석 결과로, 도 10A는 바이오 프린팅 10일 후 무 세포 양막(AM)과 리보핵산분해효소 5(RNase 5) 벡터로 형질전환된 사람 각막 내피세포(HCECs; R5-HCECs)가 가득한 양막 이식체의 투명도를 확인한 결과이며, R5-HCEC의 바이오 프린팅(무색 점선 안쪽)은 무 세포 소 양막(검은 점선 사각형)의 선명도를 감소시키지 않았다. 도 10B는 무 세포 양막 및 바이오 프린팅 10일 후 R5-이식체의 헤마톡실린 & 에오신 염색을 광학 현미경으로 확인한 결과로, HCEC의 운반체로 사용된 동결 건조된 양막에서는 세포가 관찰되지 않은 반면, 도 10Biii 및 도 10Biv와 같이 R5-이식체에서는 R5-HCEC가 양막 조직위에 25 μm 두께로 단층을 형성한 것을 확인한 결과이며 [Scale bar: 20 ㎛ (검정), 10 ㎛ (흰색)], 도 10C는 이식 전 양막 위에서 3D 바이오 프린트된 R5-HCECs를 10일간 성장시키고 소맥배아응집소(wheat germ agglutinin; WGA)로 염색하여 3D 바이오 프린트된 R5-HCECs의 세포막을 형광 현미경으로 관찰한 결과로(endothelial cell density [ECD]: 1244 cells/mm²), 접촉이 억제된 다각형 모양의 표준 세포가 사각형 안쪽에서 확인되었다 [Scale bars: 100 ㎛ (흰색), 30 ㎛ (노란색)].
도 11은 이식 가능한 3차원 바이오 프린트된 각막 내피세포 이식체의 세포질 및 표현형을 확인한 결과로, 도 11A는 바이오 프린팅 10일 후 CellTracker™ Green CMFDA 염료를 이용한 형광현미경 분석을 수행하여 이식체 상의 사람 각막 내피세포(HCECs)를 추적한 결과로, R5-HCECs 이식체(R5-Graft) 상에 리보핵산분해효소 5(RNase 5) 벡터로 형질전환된 사람 각막 내피세포(R5-HCECs)가 거의 합류되었고 다각형의 모양을 형성하였으나, 대조군 HCECs 이식체(Ct-Graft)에서는 대조군 HCECs에서는 희막하게 분포되어 있었으며, 섬유아세포 표현형을 나타내는 것을 확인한 결과이며 이미지 i 및 iii은 이미지 ii 및 iv를 확대한 것이다 [Scale bars: 500 ㎛ (흰색), 100 ㎛ (노란색)]. 도 11B는 바이오 프린팅 10일 후 R5-HCECs로 덮힌 이식체(R5-Graft)와 대조군 HCECs로 덮힌 이식체(P4; Ct-Graft)에서 Na⁺-K⁺ ATPase 및 ZO-1(zonular occludens-1)을 면역염색하여 확인한 결과로, Na⁺-K⁺ ATPase (arrow, representative in ii)의 기저 외측 발현 및 ZO-1의 결합 발현은 R5-이식체 상의 HCEC보다 Ct-이식체 상의 HCEC가 섬유아세포임을 나타낸 결과이다. 이미지 i 및 iii은 이미지 ii 및 iv를 확대한 것이며, Scale bars: 100 μm (흰색), 50 ㎛ (노란색)이다.
도 12는 3차원 바이오 프린트된 각막 내피세포 이식체 이식 후 생체 내 각막 내피 재생 변화를 시간 경과에 따라 확인한 결과로, 이식체 이식 수술 4주 후 토끼 대조군 그룹의 각막과 생체 외 조직학적 분석을 수행하여 비교한 결과이다. 도 12A는 수술 후 4주 동안 다양한 그룹에서 전방 부분을 촬영한 사진으로, 데스메막을 벗겨낸 대조군 그룹, 세포 주입 그룹 및 무 세포 양막(AM) 그룹에서는 수술 후 4주동안 중심 각막 부종 때문에 동공 여백이 가려졌으며, 세포 주입 그룹의 안구에서는 각막 혈관신생이 확인되었다(도 12ii의 검은색 화살표). 도 12A xiii 및 도 12A xviii의 검은 색 점선은 수축된 마진을 나타내며, 중심 사람 각막 내피세포(HCECs)가 삽입된 이식체(Ct-Graft) 및 리보핵산분해효소 5(RNase 5) 벡터로 형질전환된 HCECs (R5-HCECs)가 삽입된 이식체(R5-Graft) 그룹과 다르게 삽입된 무 세포 양막은 수술 3주 후 부터 수축하였다. 도 12B는 정상 토끼 각막, 데스메막이 박리된 대조군 각막 및 R5-이식체가 이식된 각막을 수술 4주 후 확인한 대표 사진으로, R5-이식체가 이식된 각막은 전방 구조 및 동공 마진이 명확하게 보일 정도로 회복된 것을 확인할 수 있었으며, 화살표 팁은 삽입된 이식체의 마진을 나타낸다. 도 12C는 대조군(n = 5 eyes), 세포 주입군(n = 3 eyes), 무 세포 양막(n = 2 eyes), Ct-이식체군(n = 5 eyes) 및 R5-이식체군(n = 6 eyes)의 중심 각막 두께(CCT)를 확인한 결과로, R5-이식체군의 각막 두께는 수술 4주 후 거의 기준치 수준으로 회복되었다. **p < 0.01 and *p < 0.05, vs. 대조군, ^# p < 0.05, vs. 세포 주입군, ^&& p < 0.01 및 ^& p < 0.05, vs. 무 세포 양막(AM) (ANOVA followed by Bonferroni's post-hoc analysis). 실험 결과값은 평균 ± s.e.m.으로 나타내었다. 도 12D는 항 염색 항체를 이용하여 형광 현미경 분석한 CellTracker™ Green CMFDA 염료 이미지이며, 도 12E는 알리자린 레드 S 염색 현미경 분석 이미지(흑백 여과)이다. 도 12D i는 양막 시트의 가장자리 안쪽에서 R5-이식체 시트 상에 추적된 세포를 확인할 수 있었으며, 도 12D ii 및 도 12E에서 세포 모양이 다각형임을 확인할 수 있었다[Scale bars: 100 ㎛ (노란색), 40 ㎛ (흰색) and 10 ㎛ (검정색)]. 도 12F는 R5-이식체 상에 R5-HCEC의 Na⁺-K⁺ ATPase, CD166 및 ZO-1(zonular occludens-1) 및 Ct-이식체 상의 대조군 HCECs의 Na⁺-K⁺ ATPase를 면역염색한 결과로, DAPI는 세포 핵 시각화를 위해 사용되었다 [Scale bars: 20 ㎛].
도 13은 RNase 5을 과발현시키기 위한 RNase 5에 대한 ORF(Gene open reading frame) cDNA 클론 발현 플라스미드(Human Angiogenin/RNase 5 gene ORF cDNA clone expression plasmid)의 개열지도이다.1 is a schematic diagram showing the process of bio-printing of human corneal endothelial cells (HCECs) cultured on the amniotic membrane.
Figure 2 is a schematic and optical picture showing the surgical process of implanting a 3D (3D) bio-printed corneal endothelial implant on the rabbit cornea, Figure 2A is a human corneal endothelial cell on the amniotic membrane after detaching the desme membrane from the rabbit cornea It is a schematic diagram for explaining the process of implanting a 3D printed implant, and FIGS. 2B to 2M are representative images of the surgical procedure, and FIG. 2B is a photograph after displaying an 8 mm circle at the center, and FIGS. 2C to 2E are A photograph showing the process of peeling and extracting a desme membrane using a reverse sinskey hook. The desme membrane extracted as shown in FIG. 2E was peeled toward the center of the cornea as shown in FIG. 2, and an 8 mm trippin blood as shown in FIG. 2F The implant cut into a circular shape with a diameter of 8 mm was folded with a blade) and inserted into the anterior chamber using a forceps for inserting an IOL (Fig. 2G). 2H and 2I, after expanding the implant, as shown in FIGS. 2J to 2M, a 2.75 mm corneal wound was sealed with # 10-0 nylon and air was injected so that the implant adhered well to the corneal stroma.
In the arrow portion of FIG. 2K, it was confirmed that the margin of the inserted implant was well attached to the inner surface of the cornea by forward air injection.
3 is a result of confirming the ribonuclease 5 (RNase 5) plasmid and control plasmid through colony PCR and transforming the cultured human corneal endothelial cells with the RNase 5 expression vector and confirming the overexpression of RNase 5, FIG. 3A Shows colony screening of DH5α cells transformed with C-green fluorescent protein (GFP) -RNase 5 plasmid and C-GFP-control plasmid, showing amplified clone 352-bp-piece of RNase 5 gene and control gene Did. Clones of lanes 6, 7, 8, 9, and 10 are recombinant, lanes 3 and 4 are identified as false positive colonies, and FIG. 3B shows PCR analysis of the RNase 5 gene in cultured human corneal endothelial cells (HCECs). results, and Fig. 3C through 3E is of 0.2 ㎍ and 0.5 ㎍ concentration RNase 5 expression vector, negative control (N / C) vector or Lipofectamine ^® 3000 in transformed human endothelial cells (HCECs) converted for 24 hours and 48 hours As a result of confirming the mRNA expression of RNase 5 in, Figure 3C is a result of confirming the RNase 5 expression level of cells transformed for 24 hours in transfection-passage 0 (t-P0), Figure 3D is transfection-passage 0 (t- P0) is the result of confirming the RNase 5 expression level of cells transformed for 48 hours, and FIG. 3E shows the level of Nase 5 expression in cells maintained for 72 hours after passage in culture medium (transfection-passage 1 [t-P1]). It is the result of confirmation.
3F is a result of confirming that endogenous RNase 5 is continuously expressed in HCECs transformed with an RNase 5 vector (0.2 μg and 0.5 μg) despite being maintained in a medium without a vector (** p <0.01, n = 4 independent experiments), the results were expressed as mean ± sem, and Western blot analysis of RNase 5 in cultured HCECs mock, N / C vector, and RNase 5 vector groups showed R5-HCECs of 12-valent t-P1 in lane 7 And β-actin was used as a loading control, and the relative concentration values for β-actin concentration in the same lane were recorded under the gel.
4 is a result of culturing human corneal endothelial cells transformed with ribonuclease 5 (RNase 5) expression vector and confirming the expression level of RNase 5 protein in cells, FIG. 4A shows transfection-passage 0 (t-P0) Representative images confirming fluorescence of green fluorescent protein (GFP) labeled RNase 5 overexpressed at C-terminus of HCECs (R5-HCECs) transformed with RNase 5 vector (0.5 μg) for 48 hours in FIGS. 4Ai to 4 Green fluorescence was observed in the nucleus 4Aiii (white arrow), and green fluorescence was observed in the cytoplasm around the nucleus in FIGS. 4Aiv to 4vi (yellow arrow). Figure 4B is a result of performing immunostaining of RNase 5 in human corneal endothelial cells (NC-HCECs) transformed with R5-HCECs and negative control (N / C) vector 0.5 μg for 48 hours, RNase 5 enriched around the nucleus. In contrast to R5-HCECs expressing or expressing (asterisk) partially enriched RNase 5 in the nucleus, NC-HCEC was found to have a rare presence of intrinsic RNase 5. FIG. 4C is a representatively enlarged image of R5-HCECs (t-P0 and t-P1), and intracellular RNase 5 coexists in the nucleus (white arrow) and the region around the nucleus (yellow arrow) connected across the nuclear membrane. The results are confirmed [(AC) Scale bars: 20 μm (white) and 200 μm (yellow)].
5 is a result of confirming the cell viability over time of human corneal endothelial cells (HCECs) transformed with ribonuclease 5 (RNase 5) expression vector and cultured, HCECs transformed with RNase 5 vector (R5- In order to compare the relative viability of HCECs with the mock group, the cell viability was confirmed by MTT analysis after maintaining in the culture medium for 6 days from the first day of transformation (D0), and FIG. 5A shows that the viability of R5-HCECs is different from the mock group. Although no difference was confirmed, in FIG. 5B and FIG. 5C, the R5-HCEC group treated with the 0.5 μg vector started to show excellent viability from D1. On days D3, 5D and 5G, the cell viability of both vector groups was better than that of the mock group [** p <0.01 and * p <0.05 (ANOVA followed by Bonferroni's post-hoc analysis), n = 6 independent experiments at all timepoints. Values represent the mean

sem].
Figure 6 is a result of confirming the effect of overexpressing ribonuclease 5 (RNase 5) in the proliferation of cultured human corneal endothelial cells, Figure 6A is HCECs transformed with RNase 5 expression vectors (R5-HCECs) compared to mock Transformation-is a result of confirming the relative amount of BrdU absorption in Fessage 1, Figure 6B is a result of confirming that the growth curve of R5-HCECs is maintained for more than 5 days in the culture medium compared to the control HCECs and mock group, of RNase 5 Overexpression significantly increased BrdU uptake (A), and results confirming that the growth rate of R5-HCECs (0.5 μg vector) was accelerated [(A) ** p <0.01. n = 4 independent experiments. (B) ** p <0.01 and * p <0.05, vs. control HCEC. ^## p <0.01 and ^# p <0.05, vs. mock. n = 5 independent experiments.].
7 is a result of confirming the expression level of micro RNA (miR) -21, miR-23a and miR-27a in human corneal endothelial cells (HCECs) transformed and cultured with ribonuclease 5 (RNase 5) expression vector. , Figures 7A to 7C is a result of confirming the expression change level of miR-21, Figure 7D to 7F is a result of confirming the expression change level of miR-23a, Figure 7G to 7I is the expression level of miR-27a As a result of confirmation, the expression level was RNase 5 expression vectors (0.2 μg and 0.5 μg, R5-HCECs) transformed with HCECs (transfection-passage 0; t-P0) transformed for 24 hours and 48 hours and culture medium for 72 hours. It is a result of quantifying the change of miRs in R5-HCECs (passage; t-P1) maintained at RT-PCR. Importantly, the up-regulation of miRs in R5-HCECs was more prominent after fertilization (t-P1) than during transfection (t-P0) [** p <0.01 and * p <0.05 (ANOVA followed by Bonferroni's post- hoc analysis). n = 3 independent experiments. Values represent the mean

sem].
FIG. 8 is a quantitative real-time RT-PCR analysis result confirming a target gene candidate of ribonuclease 5 (RNase 5) in cultured human corneal endothelial cells (HCECs) transformed with an RNase 5 expression vector, FIG. 8A shows PDCD4 ( programmed cell death protein 4; and the results confirm the level of expression of a), Fig. 8B is a result of confirming the expression level of PTEN (phosphatase and tensin homolog), Figure 8C is a result of confirming the expression level of Cdc25A, 8D are Cdk2AP1 8E is the result of confirming the expression level of Spry1 (sprouty RTK signaling antagonist 1), FIG. 8F is the result of confirming the expression level of Spry2, and FIG. 8G is Apaf1 (apoptotic protease activating factor 1) The expression level was confirmed, and the expression level is a result of comparing HCECs transformed with RNase 5 expression vector and cultured with HCECs transformed with mock group and negative control (N / C) vector [** p < 0.01 and * p <0.05 (ANOVA followed by Bonferroni's post-hoc analysis). n = 8 independent experiments. Values represent the mean

sem], FIG. 8H is a Western blot analysis result for identifying PDCD4, cyclin A2, cyclin D1, cyclin D3 and cyclin E1 in cultured mock, N / C vector and RNase 5 vector HCECs group, lanes 7 to 12 are t R5-HCECs of -P1, β-actin was used as a loading control, and relative concentration values for β-actin density were recorded under each gel in the same lane.
9 is a result of confirming the expression level of a function-related marker in human corneal endothelial cells (HCEC) and control HCECs cultured by transformation with a ribonuclease 5 (RNase 5) expression vector, and FIG. 9A is a 20-year-old female donor ( Primary HCECs (passage 0; P0) cultured from A) are confirmed by phase contrast microscopy, and FIG. 9B shows CD166 and CD44 expression levels before and after RNase 5 vector transformation, and the HCEC cell population is CD166 and CD44 positive. In addition, the surface marker is also a result of flow cytometry analysis of HCECs (R5-HCECs) transformed with 0.5 µg of the RNase 5 vector, which was also positive, for 48 hours at t-P1 (transfection-passage 1) (Scale bar: 100 μm). 9C is a result of phase contrast microscopy analysis of control HCECs (P4) and R5-HCECs (t-P1) and immunostaining results confirming Na ⁺ -K ⁺ ATPase and zonular occludens-1 (ZO-1) of the cell group. Both the fusion R5-HCECs and control HCECs of 9Ci and FIG. 9Cii show relatively fibroblast phenotypes, but the polygonal cell morphology and tight junctions between cells were more confirmed in R5-HCEC than in control HCEC. Na ⁺ -K ⁺ ATPase expression was found in the lateral membrane of the cell and was significantly higher in R5-HCECs than in HCECs. In addition, as shown in FIG. 9Cx, some of these shapes were confirmed as a standard form according to ZO-1 expression at a tight intersection (white dotted border). Images iv, vi, viii and x are enlarged images iii, v, vii and ix, respectively, and DAPI was used for cell nuclear visualization [Scale bars: 100 μm (white), 20 μm (yellow)]. 9D is a real-time RT-PCR analysis result quantitatively confirming ATP1A1 (ATPase Na ⁺ -K ⁺ transporting subunit alpha 1) gene expression, and FIG. 9E is a real-time RT-PCR quantitatively confirming TJP1 (tight junction protein 1) gene expression. 9F is a real-time RT-PCR analysis result quantitatively confirming ALCAM (activated leukocyte cell adhesion molecule; ie CD166) gene expression, and FIG. 9G is a real-time RT-PCR analysis result quantitatively confirming CD44 gene expression. The results of confirming the gene expression amount are the results of comparing the gene amount expressed in R5-HCECs with HCECs transformed with a mock group and a negative control vector. [** p <0.01 and * p <0.05 (ANOVA followed by Bonferroni's post-hoc analysis). n = 8 independent experiments. Values represent the mean

sem]. 9H is a Western blot analysis result confirming Na ⁺ -K ⁺ ATPase, ZO-1, C166 and CD44 expression in HCECs cells transformed with mock, N / C vector and RNase 5 vector, β-actin as a loading control. The relative concentration values for β-actin density in the same lane were recorded under each gel.
FIG. 10 shows the results of the clarity and histological analysis of a cell-free amniotic membrane (AM) and implantable 3-dimensional (3D) bio-printed corneal endothelial implant, and FIG. This is a result of confirming the transparency of the amniotic membrane implanter full of human corneal endothelial cells (HCECs; R5-HCECs) transformed with RNase 5 vector, and bioprinting of R5-HCEC (inside the colorless dotted line) is cell-free. The clarity of the amniotic membrane (black dotted rectangle) was not reduced. Figure 10B is a result of confirming hematoxylin & eosin staining of R5-grafts 10 days after cell-free amniotic membrane and bioprinting, while cells were not observed in the freeze-dried amniotic membrane used as a carrier of HCEC. As shown in 10Biii and 10Biv, in the R5-graft, it was confirmed that R5-HCEC formed a monolayer with a thickness of 25 μm on the amnion tissue. [Scale bar: 20 μm (black), 10 μm (white)], FIG. 10C 3D bioprinted R5-HCECs were grown on the amniotic membrane for 10 days prior to transplantation, stained with wheat germ agglutinin (WGA), and the cell membrane of 3D bioprinted R5-HCECs was observed with a fluorescence microscope (endothelial cell density [ECD]: 1244 cells / mm ² ), standard cells of polygonal shape with inhibited contact were identified inside the square [Scale bars: 100 μm (white), 30 μm (yellow)].
11 is a result of confirming the cytoplasm and phenotype of a transplantable 3D bio-printed corneal endothelial cell transplant, and FIG. 11A shows fluorescence microscopy analysis using CellTracker ™ Green CMFDA dye 10 days after bioprinting to human cornea on the implant. As a result of tracking endothelial cells (HCECs), human corneal endothelial cells (R5-HCECs) transformed with the ribonuclease 5 (RNase 5) vector on the R5-HCECs transplant (R5-Graft) were almost joined. Although the shape of the polygon was formed, the control HCECs implant (Ct-Graft) was found to be rarely distributed in the control HCECs, and it was confirmed that the fibroblast phenotype was shown. Images i and iii are enlarged images ii and iv [Scale bars: 500 μm (white), 100 μm (yellow)]. FIG. 11B shows Na ⁺ -K ⁺ ATPase and zonular occludens-1 in Na ⁺ -K ⁺ ATPase (P4; Ct-Graft) covered with R5-HCECs after 10 days of bioprinting (R5-Graft) and control HCECs. As a result of immunostaining), Na ⁺ -K ⁺ ATPase (arrow, representative in ii) of basal lateral expression and binding expression of ZO-1 showed that the HCEC on the Ct-graft is more fibroblast than the HCEC on the R5-graft. This is the result. Images i and iii are enlarged images ii and iv, Scale bars: 100 μm (white), 50 μm (yellow).
12 is a result of confirming changes in corneal endothelial regeneration in vivo after transplantation of a 3D bio-printed corneal endothelial cell transplant, and performed corneal and in vitro histological analysis of the rabbit control group 4 weeks after the transplantation surgery. This is the result of comparison. 12A is a photograph of the anterior portion taken in various groups for 4 weeks after surgery. In the control group, the cell injection group, and the cell-free amniotic membrane (AM) group, in which the desme membrane was peeled off, pupil margin due to central corneal edema for 4 weeks after surgery. This was masked, and corneal angiogenesis was confirmed in the eye of the cell injection group (black arrow in FIG. 12ii). The black dashed lines in FIGS. 12A xiii and 12A xviii represent contracted margins, and are transformed with central human corneal endothelial cells (HCECs) inserted implants (Ct-Graft) and ribonuclease 5 (RNase 5) vector. The cell-free amniotic membrane inserted differently than the implanted group (R5-Graft) with inserted HCECs (R5-HCECs) contracted after 3 weeks of surgery. FIG. 12B is a representative photograph confirming the normal rabbit cornea, the control cornea from which the Desme membrane was detached, and the cornea transplanted with the R5-graft 4 weeks after surgery, and the cornea implanted with the R5-graft is clearly visible in anterior structure and pupil margin. The recovery was confirmed, and the arrow tip indicates the margin of the inserted implant. 12C shows control group ( n = 5 eyes), cell injection group ( n = 3 eyes), cell-free amniotic membrane ( n = 2 eyes), Ct-graft group ( n = 5 eyes) and R5-graft group ( n = 6) As a result of confirming the central corneal thickness (CCT) of the eyes), the corneal thickness of the R5-graft group recovered almost to the baseline level after 4 weeks of surgery. ** p <0.01 and * p <0.05, vs. Control, ^# p <0.05, vs. Cell infusion group, ^&& p <0.01 and ^& p <0.05, vs. Cell-free amniotic membrane (AM) (ANOVA followed by Bonferroni's post-hoc analysis). The experimental results were expressed as the mean ± sem. 12D is an image of CellTracker ™ Green CMFDA dye analyzed by fluorescence microscopy using an anti-staining antibody, and FIG. 12E is an alizarin red S staining microscopy image (monochrome filtration). 12D i was able to confirm the traced cells on the R5-graft sheet inside the edge of the amnion sheet, and it was confirmed that the cell shape was polygonal in FIGS. 12D ii and 12E [Scale bars: 100 μm (yellow), 40 μm (white) and 10 μm (black)]. Figure 12F is R5- of R5-HCEC on the implant Na ⁺ -K ⁺ ATPase, CD166, and ZO-1 (zonular occludens-1 ) and one control Ct- implant immunostaining for the Na ⁺ -K ⁺ ATPase HCECs on As a result, DAPI was used for cell nuclear visualization [Scale bars: 20 μm].
13 is a cleavage map of a human open reading frame (ORF) cDNA clone expression plasmid for RNase 5 to overexpress RNase 5 (Human Angiogenin / RNase 5 gene ORF cDNA clone expression plasmid).

Hereinafter, the present invention will be described in more detail.

The present invention is a step of overexpressing ribonuclease 5 (Ribonuclease 5) on corneal endothelial cells (first step); Dispersing corneal endothelial cells overexpressed in the first step of ribonuclease 5 in bioink (second step); Separating the bioink in which the corneal endothelial cells of the second step are dispersed from livestock and bioprinting the lyophilized amniotic membrane (third step); And it may provide a three-dimensional bio-printed corneal endothelial implant manufacturing method comprising the step of cross-linking by irradiating ultraviolet rays to the bio-printed amniotic membrane of the third step (step 4).

Corneal endothelial cells in which the ribonuclease 5 of step 1 is overexpressed is transformed into corneal endothelial cells with a recombinant vector containing the ribonuclease 5 gene encoding the amino acid sequence represented by SEQ ID NO: 1 to transform the ribonucleic acid. It may be that the enzyme 5 is overexpressed.

The recombinant vector may have a cleavage map shown in FIG. 13, and the corneal endothelial cells may be corneal endothelial cells isolated from humans.

The bioink in which the corneal endothelial cells in the second step are dispersed may be to disperse the corneal endothelial cells in which the ribonuclease 5 is overexpressed in a bio ink at a concentration of 3 × 10 ⁶ cells / mL.

The third step may be to bioprint the bio-ink in which corneal endothelial cells are dispersed to a small amniotic membrane with a thickness of 0.5 to 1 mm.

More specifically, the bovine amniotic membrane may be lyophilized and acellularized by chemical enzyme treatment.

The fourth step may be to cross-link the bioprinted amniotic membrane by irradiating 360 nm ultraviolet rays for 10 to 20 seconds.

The method of manufacturing the corneal endothelial implant may further include a step of culturing the corneal endothelial implant irradiated with ultraviolet rays after the fourth step at 37 ° C for 10 days.

In addition, the present invention can provide a corneal endothelial implant in which a corneal endothelial cell overexpressing ribonuclease 5 according to the method for preparing the corneal endothelial implant is 3D bioprinted on the amniotic membrane.

The corneal endothelial implant may have increased expression of Na ⁺ -K ⁺ ATPase and CD166 in corneal endothelial cells overexpressing 3D bioprinted ribonuclease 5 on the amniotic membrane.

The corneal endothelial implant may have increased corneal endothelial phenotype due to increased expression of zonular occludens-1 (ZO-1) in corneal endothelial cells overexpressing 3D bioprinted ribonuclease 5 on the amniotic membrane. have.

According to an embodiment of the present invention, the rabbit eye was a control group (5 eyes), a cell infusion group (3 eyes), a cell-free AM group (2 eyes), a Ct-graft group (5 eyes), and an R5-graft group ( 7 eyes), and after removing the desmem of the eye cornea of each experimental group rabbit with a circular shape of 8.0 mm diameter, the round implant (R5-graft, control-graft or cell-free small membrane) was described as DSEK (Descemet's Stripping Endothelial Keratoplasty) ). In addition, R5-HCEC cells cultured in additional groups were injected into the anterior chamber, and all these experimental groups were compared to a group (control) in which no manipulations such as implantation of implants or cell implantation were performed after desmemak separation.

As a result of histological observation and analysis of corneal endothelium in 3 eyes of the R5-graft group 4 weeks after surgery, the rear part of the transplanted R5-graft was filled with a single layer of cells with no gaps, and the average density of the cells was 3,073 ± 341.2 (Mean ± standard error) It was confirmed as cells / mm ² , and anti-dye fluorescence was observed under a microscope. As shown in FIG. 12E, these were not corneal endothelial cells of rabbits, but human corneal endothelial cells cultured and transplanted. Corneal endothelial cells on the surface of the transplanted R5-graft surface clearly expressed markers such as Na ⁺ -K ⁺ ATPase, ZO-1 and CD166 along the hexagonal border of the cells, whereas Na in the control-graft corneal endothelial cells ⁺ -K ⁺ ATPase expression was relatively low compared to R5-graft.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Reference example > Substances, reagents and antibodies

Materials, reagents and antibodies used in the present invention are as follows: human recombinant RNase 5 (synonym for ANG) protein (# 265-AN / CF, R & D Systems, Minneapolis, MN, USA), microRNA primer: (miR)- 21 (# MS00009079, miScript Primer Assays, QIAGEN, Hilden, Germany), miR-23a (# MS00031633, QIAGEN), miR-27a (# MS00003241, QIAGEN); Abs: mouse monoclonal Abs (mAbs) anti-RNase 5 (# 14017.7, Abcam, Cambridge, MA, USA), anti-Na ⁺ / K ⁺ ATPase (# 464.6, Abcam), anti-cyclin E1 (# 4129, Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (# 5441, Sigma-Aldrich, St. Louis, MO, USA); rabbit mAbs: anti-cyclin D1 (# 2978, Cell Signaling Technology) and anti-programmed cell death (PDCD) 4 (# 9535, Cell Signaling Technology); rabbit polyclonal Abs (pAbs): anti-zonula occludens (ZO) -1 (# 40-2200, Invitrogen, Waltham, MA, USA), anti-CD44 (# HPA005785, Atlas Antibodies AB, Stockholm, Sweden), anti-CD166 (# 109215, Abcam), anti-cyclin-dependent kinase (Cdk) 2 (# A301-812A-T, Bethyl Laboratories, Montgomery, TX, USA), anti-Cdk4 (# A304-225A, Bethyl Laboratories) and anti- cyclin A2 (# A305-253A, Bethyl Laboratories) antibody was used, and the materials and reagents were Opti-MEM-I (# 31985070, Gibco®, Life Technologies Inc.TM, Carlsbad, CA, USA), fetal bovine serum (FBS , # S001-01, Welgene, Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea), chondroitin sulfate (# C9819-5g, Sigma-Aldrich), ascorbic acid (# A5960-25g, Sigma- Aldrich), CaCl ₂ (# C5670-100g, Sigma-Aldrich), multivitamin solution (RPMI 1640 vitamins solution, # R7256, Sigma-Aldrich), gentamicin (# 15750060, Gibco®), penicillin / streptomycin antibiotic Gland (PSA, # 15140122, Gibco®), pituitary extract (# 13028014, Gibco®), epidermal growth factor (EGF, # E9644, Sigma-Aldrich) and nerve growth factor (NGF, # 450-01, PeproTech, Rocky Hill, NJ, USA).

< Experimental Example  1> People Corneal endothelial cells (Human Corneal Endothelial Cells; HCECs ) Culture

After transplanting by incising the central 8 mm round cornea, the remaining peripheral corneal tissue was cultured to separate human corneal endothelial cells (HCECs).

Human corneal tissue was obtained from 9 donors (13 eyes) without any ocular disease, and 2 eyes (# 1, 2; primary cause of death [PCOD]: brain haemorrhage; endothelial cell density [ECD] from a 52-year-old woman) : 3,076 and 2,570 cells / mm ² ) and 2 eyes from a 29-year-old male (# 3, 4; PCOD: brain haemorrhage; ECD: 2,687 and 3,373 cells / mm ² )

Two eyes (# 5, 6; PCOD: brain haemorrhage; ECD: 2,886 and 2,887 cells / mm ² ) were donated from a 40-year-old male, and two eyes (# 7, 8; PCOD: brain haemorrhage) from a 54-year-old male. ; ECD: 2,986 and 2,912 cells / mm ² ).

One eyeball (# 9; PCOD: brain haemorrhage; ECD: 3,252 cells / mm ² ) from a 20-year-old woman, and one eyeball (# 10; PCOD: pulmonary fibrosis; ECD: from a 66-year-old male) 2,342 cells / mm ² ) was purchased Mid-America Transplant (St. Louis, MO, USA).

In addition, one eye (# 11; PCOD: myocardial infarction; ECD: 2,532 cells / mm ² ) donated to a 57-year-old male was purchased from Lone Star Lions Eye Bank (Manor, TX, USA).

One eyeball (# 12; PCOD: acute cardiac event; ECD: 2,639 cells / mm2) from a 63-year-old woman was purchased from Lone Star Lions Eye Bank and one eyeball from a 18-year-old male (# 13 PCOD: cardiomyopathy; ECD: 3,436 cells / mm2) was purchased from Cincinnati Eye Bank (Cincinnati, OH, USA).

All procedures were based on previously known methods (Kim KW, Park SH, Lee SJ, Kim JC. Ribonuclease 5 facilitates corneal endothelial wound healing via activation of PI3-kinase / Akt pathway.Sci Rep . 2016; 6: 31162.).

Human corneal endothelial cells (HCECs) obtained from incised corneal-sclerotic rings after corneal transplantation were stored in storage medium (Optisol-GS, Bausch and Lomb, Inc., Rochester, NY, USA) until the experiment.

HCECs have been reported previously (Kim E, Kim JJ, Hyon JY, et al. The effects of different culture media on human corneal endothelial cells.Invest Ophthalmol Vis Sci . 2014; 55: 5099-5108.) 8% FBS, 0.08% chondroitin sulfate, ascorbic acid 20 μg / mL, CaCl ₂ 200 μg / mL, multivitamin solution (1: 100), pituitary extract (1: 100) Maintained in fresh medium consisting of reduced serum medium (Opti-MEM-I) containing 100 μg / mL, EGF 5 ng / mL, NGF 20 ng / mL, gentamicin 50 ng / mL and PSA (1: 100) Became.

Briefly, using sterile surgical forceps, carefully desmec sheet with intact endothelial cells and carefully incubated with collagenase A 1 mg / mL for 2 hours at 37 ° C. for 5 minutes at 1,200 rpm. HCECs were centrifuged.

The supernatant was removed, and the HCECs were washed with a phosphate-buffered saline (PBS) and re-centrifuged at 1,200 rpm for 5 minutes.

Then, corneal endothelial cells were adhered to a tissue culture dish coated with fibronectin, and then the separated cells were cultured in the medium.

When human corneal endothelial cells showed 80-90% confluence, they were separated using trypsin / EDTA and dispensed at a density of 10,000 cells / cm ² to be passaged.

All incubation and cultivation of human corneal endothelial cells was performed in a 37 ° C. and 5% CO ₂ humidified incubator and replaced every other day with fresh medium.

< Experimental Example  2> RNase  People transformed with 5 expression vectors Corneal endothelial cells  making

Gene open reading frame (ORF) cDNA clone expression plasmid for RNase 5 (Human Angiogenin / RNase 5 gene ORF cDNA clone expression plasmid: (1) C-green fluorescent protein (GFP) Spark tag [# HG10441-ACG, Sino Biological Inc ., Beijing, China] or as a negative control vector (2) C-Flag tag [# HG10441-MF, Sino Biological Inc.]) or pCMV3-C-GFPSpark Control Vector (C-terminal GFP Spark-tagged, # CV026, Sino Biological Inc.) was transformed into E. coli according to the manufacturer's instructions (Protocols 2 in Rapid MacCell ™ -DH5α, # 15062, iNtRON Biotechnology Inc., Gyeonggi-do, Republic of Korea).

To propagate vector transformed colonies, the cells were cultured by stirring at 230 rpm overnight at 37 ° C. in a conical tube containing 4 mL of LB (lysogeny broth) medium containing 100 μg / mL of kanamycin.

Plasmid preparation was performed according to the manufacturer's instructions [HiGene ™ Plasmid Mini Prep Kit (# PM119, BIOFACT, Daejeon, Republic of Korea)].

Subsequently, 0.2 and 0.5 μg of RNase 5 expression vector or N / C vector according to the manufacturer's instructions using Lipofectamine ^® 3000 transfection reagent (7.5 μL) (# L3000008, Invitrogen) in HCECs cultured to be confluent to 70 to 80%. Transformed to concentration for 72 hours.

HCECs cultured to overexpress RNase 5 after transformation with RNase 5 expression vector were named R5-HCECs, and HCECs cultured with N / C vector transformation were named NC-HCECs.

The cell group treated with Lipofectamine® 30 million in HCECs was selected as a mock group, and real-time RT-PCR (qRT-PCR) was performed to confirm the RNase 5 overexpression efficiency.

< Example  3> Cell growth analysis

The cultured HCECs were inoculated to a 35-mm dish plate (SPL life sciences, Pocheon, Gyeonggi-do, Republic of Korea) at a density of 1 × 10 ⁵ cells / mL and cultured in the medium for 1 to 5 days.

The medium was then removed and the cells were trypsinized. The number of HCECs was checked using a hemocytometer for 5 days after trypan blue staining.

5 RNase-overexpressing vector and then (0.2 ㎍ and 0.5 ㎍) to Lipofectamine ^® 3000 (7.5 μL) or RNase 5 expression vector Lipofectamine® 3000 (7.5 μL) not containing a treatment for 48 hours in HCECs, subcultured in the culture medium Cells were maintained in the culture medium for 24 hours before the start of the experiment (D0).

< Experimental Example  4> BrdU  Cell proliferation assay

Cell proliferation was confirmed according to the manufacturer's instructions using the BrdU Cell Proliferation Assay Kit (# 6813, Cell Signaling Technology).

The HCECs to a 96 well plate 0.5 × 10 ⁴ cells / well density divided by the RNase 5 overexpression vector (0.2 ㎍ and 0.5 ㎍) with Lipofectamine ^® 3000 (7.5 μL) or RNase-free vector 5 expression Lipofectamine® 3000 contain (7.5 μL) was treated with HCECs for 72 hours.

Thereafter, a 10x BrdU solution was added to each well of the plate to reach a final 1x concentration and treated for 24 hours.

HCECs were immobilized and then incubated with mouse anti-BrdU antibody (Ab) followed by peroxidase bound anti-mouse Ab.

The reaction was stopped by adding 3,3 ', 5,5'-tetramethylbenzidine peroxidase substrate to express color, incubating for 30 minutes, and then adding a stop solution.

BrdU binding levels were confirmed at 450 nm using a SpectramaxTM 340PC384 microplate photometer (Molecular Devices, Sunnyvale, CA, USA).

< Experimental Example  5> Cell viability ( MTT ) analysis

After incubating HCECs in 96-well culture plates and treating Lipofectamine® 3000 with RNase 5 overexpression vectors (0.2 μg and 0.5 μg) or Lipofectamine® 3000 without RNase 5 expression vectors (7.5 μL) with HCECs for 48 hours, Starting at D0, it was passaged with culture medium.

Cell viability was confirmed using the MTT assay.

Briefly, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) was dissolved in PBS at a concentration of 5 mg / mL. MTT was added to each well at 10 μL per 100 μL of medium and incubated for 2 hours at 37 ° C. and dark conditions.

Then, the medium was replaced with 100 μL of DMSO (dimethyl sulfoxide), and the absorbance of each well was measured at 570 nm using a SpectramaxTM 340PC384 microplate photometer.

< Experimental Example  6> Real Time PCR

RNA was isolated according to the manufacturer's instructions using RNAiso plus (Takara Bio, Inc., Otsu, Japan).

For qRT-PCR analysis, total RNA is reverse transcribed into complementary DNA (PrimeScript ™ 1st strand cDNA synthesis kit for mRNA, # 6110A, Takara Bio, Inc.), and miScript® II RT kit (# 218161, QIAGEN) is used. The miRNA was reverse transcribed.

mRNA was performed using SYBR Premix ExTaq ™ (# RR420A, Takara Bio, Inc.), and miRNA was QRT-PCR using miScript SYBR® Green PCR Kit (# 218075, QIAGEN).

SybrGreen fluorescence of the amplified cDNA product was quantified using an appropriate standard curve obtained from a CFX96 Real-Time PCR Detection System (BioRad, Hercules, CA, USA) and an autonomous qPCR analysis reaction.

After standardization with a reference gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH] for mRNA and U6 small nuclear RNA [RNU6-2, # MS00033740, QIAGEN] for miRNA), the relative gene weight is determined using a comparative cycle threshold (Ct) method. The results of the qRT-PCR analysis showed the expression level of each gene as a relative value to the reference gene expression average.

Table 1, RNase 5, CD44, CD166, ATP1A1 (sodium / potassium-transporting ATPase subunit alpha-1), TJP1 (tight junction protein 1), Cdc25A (cell division cycle 25A), Cdk2AP1 (cyclin-dependent kinase 2-associated qRT-PCR analysis was performed using protein 1), PDCD4, PTEN (phosphatase and tensin homolog), Spry1 (sprouty homolog 1), Spry2, Apaf1 (apoptotic protease activating factor 1), and GAPDH gene specific primers.

Gene Primer Sequence
Product size ( bp) Reference number RNase  5 Sense 5'-TTGCGTTTTCTACCGGCTCC-3 ' 231 NM_001145.4 Antisense 5'-CAGGAAGTGTGTGTACCTGGA-3 ' CD44 Sense 5'-GTGATGGCACCCGCTATG-3 ' 179 NM_000610.3 Antisense 5'-ACTGTCTTCGTCTGGGATGG-3 ' ALCAM  (CD166) Sense 5'-CCCCAGAGGAATTTTTGTTTTAC-3 ' 289 NM_001243281.1 Antisense 5'-AGCCTGATGTTATCTTTCATCCA-3 ' ATP1A1  (Na ⁺ -K ⁺  ATPase) Sense 5'-CAGCACGCAGGTTGCATATT-3 ' 220 NM_000701.7 Antisense 5'-GAGCCAAGTGGAGGGAGCTA-3 ' TJP1
( ZO -One) Sense 5'-GAACGAGGCATCATCCCTAA-3 ' 218 NM_001330239.1 Antisense 5'-CCAGCTTCTCGAAGAACCAC-3 ' Cdc25A Sense 5'-ACCTCAGAAGCTGTTGGGATG-3 ' 174 NM_001789.2 Antisense 5'-TGGAGTCCATGAGAGTGCAG-3 ' Cdk2AP1 Sense 5'-CAGACCTTACAAAGACGGGCT-3 ' 221 NM_001270433.1 Antisense 5'-GCGTACGTGGGTCTGATCTC-3 ' PDCD4 Sense 5'-ACCCTGCAGATCCTGATAACT-3 ' 151 NM_014456.4 Antisense 5'-CGCCTTTTTGCCTTGGCATT-3 ' PTEN Sense 5'-TCCCAGACATGACAGCCATC-3 ' 189 NM_000314.6 Antisense 5'-GCTTTGAATCCAAAAACCTTACTAC-3 ' Spry1 Sense 5'-GTGTGTTGGAAATCCACGGT-3 ' 171 NM_005841.2 Antisense 5'-AAAGAAGGCTGCTGGATCAC-3 ' Spry2 Sense 5'-AGATCAGAGCCATCCGAAACA-3 ' 170 NM_001318536.1 Antisense 5'-AGAATGGACCTGCGAGTGC-3 ' Apaf1 Sense 5'-ATGAGCCCACTCAACAGCAA-3 ' 170 NM_001160.2 Antisense 5'-TGTCCTTACACTGGAAGAAGAGA-3 ' GAPDH Sense 5'-TGATGACATCAAGAAGGTGGTGAAG-3 ' 240 NM_002046.5 Antisense 5'-TCCTTGGAGGCCATGTGGGCCAT-3 '

< Experimental Example  7> Western Blot

Cells were washed twice with PBS and lysed with PRO-PREP ™ (iNtRON Biotechnology Inc.). Cell lysates were separated on sodium dodecyl sulfate (SDS) -polyacrylamide gel and transferred to a nitrocellulose (NC) membrane (Pall Corporation, Port Washington, NY, USA).

Non-specific antibody at room temperature for 1 hour with Tris buffered saline (TBS-T; 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Tween-20) containing 3% bovine serum albumin (BSA) The binding was blocked, and the primary antibody was diluted (1: 1,000 except RNase 5, which was 1: 250) with TBS-T containing 5% BSA and applied to the membrane and incubated overnight at 4 ° C.

The appropriate secondary antibody was diluted 1: 3,000 with TBS-T containing 5% skim milk and then incubated for 1 hour at membrane and room temperature, and enhanced chemiluminescence Western Blotting detection kit (Pierce Biotechnology, Inc., Rockford, IL, USA ) To visualize the binding of specific antibodies.

The values of each band are normalized to β-actin signals and Image J software ver. Immune bands were quantified using 1.46 (National Institutes of Health (NIH); http://rsbweb.nih.gov/ij/).

< Experimental Example  8> Fluorescence Active  Cell selection ( FACS )

HCECs were inoculated in a 6-well plate at a concentration of 1 × 10 ⁵ cells / mL, and after 48 hours, cells were collected and washed with cold PBS.

5 x 10 ⁵ cells were dispensed into each tube, PBS containing 10% BSA as a blocking solution buffer was added to each tube, and the cells were incubated on ice for 30 minutes.

For direct immunofluorescence, primary mouse monoantibody (mAbs) anti-CD166-PE (# A22361, Beckman Coulter, Inc., Brea, CA, USA), anti-CD44-FITC (# IM1219U, Immunotec , Inc., Vaudreuil-Dorion, Canada) or the respective isotype control single antibody mouse IgG1-PE (# A07796, Beckman Coulter, Inc.), mouse IgG1-FITC (# A07795, Beckman Coulter, Inc.) and mouse IgG1 The isotype control (# 02-6100, Invitrogen) was suspended for 5 min at 5 μg / mL in HCECs and 4 ° C. in dark conditions, and then fixed with cold 2% paraformaldehyde (PFA) at 4 ° C. for 20 min, flow cytometer ( Navios, Beckman Coulter).

< Experimental Example  9> 3D Bioprinted HCEC Implant  Manufacturing method

The bioprinting was performed in the same process as in FIG. 1 to prepare an implant.

HCECs cultured before three-dimensional (3D) bioprinting were tracked by treatment with 5 μM of CellTracker ™ Green CMFDA (5-chloromethylfluorescein diacetate; # C2925, Invitrogen) for 30 minutes.

To prepare a cell-filled bioink solution, HCECs were added to 3 × 10 ⁶ cells / in gelatin-based bioink (Gel4Cell®, Bioink Solutions, Inc., Daegu, South Korea), an optimal form of gelatin-based hydrogel complex. After dispersing to a mL concentration, 0.02% Arginylglycylaspartic acid (RGD) was added to the bioink.

The solution containing the cells was transferred to a fine nozzle tip (24 gauge) and stored on ice for a non-printing time. Lyophilized bovine amniotic membrane (AM; Amnisite®-BA, SK bioland, Cheongju-si, Chungcheongbuk-do, Republic of Korea) was placed on a 60 mm cell culture dish with a size of 10 × 10 mm ² (150 to 200 μL per sheet).

During bio-printing, the bio-ink was made of a large porous structure in the form of a stacked layer of 8 × 8 mm and 0.7 mm thick with a 3D printing system (EDISON INVIVO, ROKIT, Seoul, Republic of Korea).

After crosslinking with 365 nm UV (UV) for 15 seconds, staining of living cells was performed in several implants to confirm that HCECs were uniformly and densely supplied onto the amniotic membrane (AM).

In order to increase the number of HCECs on the amniotic membrane structure before transplantation, the prepared implants were cultured for 10 days at 37 ° C. and 5% CO ₂ in a culture medium with reduced FBS (2%).

The R5-HCECs transformed with the RNase 5 vector (0.5 μg) for 48 hours before printing were passage cultured and maintained in the culture medium for 72 hours, and then the 3D bio-printed AM construct was transferred to the R5-graft group. The 3D printed construct with control HCECs of passage 4 (P4) was defined as the Ct-Graft group.

< Experimental Example  10> Live cell staining

Two-color fluorescence analysis (Molecular ProbesTM LIVE / DEADTM assays consisting of calcein as a marker of viable cells and ethidium homodimer as a marker of dead cells, Invitrogen) was performed to check cell viability in contrast.

Each sample was washed 3 times with PBS before staining, stained for 15 minutes in dark condition, and then washed 3 times with PBS. Thereafter, an image was captured using an inverted microscope (IX71, Olympus, Tokyo, Japan).

< Experimental Example  11> Implant  Histological evaluation

Implants preserved in culture medium for 10 days prior to transplantation were fixed overnight with 4% PFA and then unfrozen overnight with 30% sucrose. Then, it was put in an optimal cutting temperature (OCT) compound (Tissue-Tek® O.C.T.Compound, Sakura Finetek USA, Inc., Torrance, CA, USA) at -80 ° C dry ice.

The portion of the frozen OCT compound was cut to a thickness of 6 μm and placed on a microscope slide coated with silane. Stained with hematoxylin-eosin and observed with an optical microscope (Leica DM750, Leica Microsystems Ltd., Wetzlar, Germany).

Plasma membranes with Wheat Germ Agglutinin (WGA) conjugates (WGA, Alexa Fluor® 488 conjugate, # W11261, Invitrogen), according to the manufacturer's instructions, to determine the boundaries of HCECs inoculated on the implant sheet prior to transplantation The image was documented using an inverted microscope (IX71, Olympus).

< Experimental Example  12> Animal Experiment

1. Experimental animals

Twelve New Zealand white rabbits (24 eyes; 10 to 12 weeks old female, 2.0 to 2.5 kg) were bred at the Clinic Research Center and used for research at Chung-Ang University.

The rabbit eye was divided into five groups:

The control group (5 eyes), cell infusion group (3 eyes), cell-free AM group (2 eyes), Ct-graft group (5 eyes) and R5-graft group (7 eyes) and both eyes confirmed normal corneal confirmation. Was used.

2. Surgery process

For anesthesia, a mixture of tiletamine and zolazepam (ZoletilTM, Virbac, Fort Worth, TX, USA; 0.2 cc / Kg) and xylazine (RompunTM, Bayer, Leverkusen, Germany; 0.1 cc / Kg) was injected intramuscularly.

After marking a 8.0 mm circle on the corneal surface with a pen, a 2.75 mm corneal limbal incision is made with a slit knife and the anterior chamber (cohesive ocular viscoelastic device, OVD; ProviscTM, Alcon, Fort Worth, TX, USA) ).

Subsequently, a corneal endothelial disorder was induced by peeling 8 mm of the center of the desmemak using a reverse Sinskey hook (customized order from BELLEIF, Suzhou, China).

After removing the OVD injected into the anterior chamber, 8 mm trephine blade (# E3096 8.00, Storz, El Segundo, CA, USA) was added, and then a 3D bioprinted corneal endothelial implant was cut and folded. The Thornton intraocular lens forcep (# E2988, Storz) was used to insert the HCEC-inoculated side facing the anterior chamber.

After the implant was unfolded, a 2.75 mm corneal wound was closed, discontinuously closed with # 10-0 nylon, and air was injected to attach the implant sheet to the area where the desmem film was peeled off.

Immediately after attachment, levofloxacin ophthalmic eyedrops (CravitTM, Santen Pharmaceutical Co., Ltd., Osaka, Japan) and antibiotic and steroid mixed ointments (MaxitrolTM, Alcon) were added.

In the cell-injected eye group, R5-HCECs (5 × 10 ⁵ cells in 50 μL of PBS) transformed with the RNase 5 vector directed toward the corneal endothelium were injected directly into the anterior chamber, and the desmemak was peeled off by gravity. The eyes were immediately closed and maintained for 1 hour so that the cells injected into the cells could be deposited.

The preliminary surgical procedure was performed using a stereomicroscope (Leica M60, Leica Microsystems Ltd.), and ophthalmic ointment (MaxitrolTM) was applied to all animal eyes once a day for 4 weeks after surgery. No systemic immunosuppressant was used, and the description of the surgical procedure and the optical image are shown in FIG. 2.

< Experimental Example  13> Check the thickness of the central cornea

Corneal edema was assessed by central corneal thickness (CCT), and central corneal thickness (CCT) was measured by ultrasound corneal pachymeter (POKET-II, Quantel medical, Clemont-Ferrand, rabbit cornea 1, 2, 3, and 4 weeks after surgery). France).

The instrument can measure corneal thickness up to 1,000 μm, and the unpredictable value of CCT above the limit was considered 1,000 μm for statistical analysis.

For analysis, three repeat measurements and averages were recorded.

< Experimental Example  14> In vitro corneal endothelial cell identification

4 weeks after surgery, corneal tissue was dissected and infiltrated with 1% alizarin red S solution (Lab Chem, Pittsburgh, PA, USA) and washed with PBS after 2 minutes.

Thereafter, the tissue was placed flat on a glass slide so that the endothelial side was visible and photographed using an inverted microscope (IX71, Olympus).

To increase the fluorescence of CMFDA initially traced in HCECs cultured 4 weeks after surgery, an anti-fluorescein / Oregon GreenTM polyclonal antibody, Alexa FluorTM 488 conjugate, # A-11096, Invitrogen was used.

After corneal tissue incision, it was fixed overnight with 4% paraformaldehyde and anti-stained antibody (1: 200) was treated for 24 hours prior to photo documentation.

To perform an immunocytochemical method of ex vivo corneal tissue 4 weeks after surgery, non-specific binding was blocked for 1 hour at room temperature using PBS containing 10% normal goat blocking solution, and overnight. Anti-Na + -K + ATPase antibody (Ab), anti-ZO-1 (1: 100) or anti-CD166 (1) diluted 1: 100 in PBS with 1% normal goat serum and 0.1% Triton X-100 : 100). Thereafter, the slides were incubated with the secondary antibody for 2 hours at room temperature.

The endothelial layer was placed flat on a glass slide and fixed with a Fluoroshield ™ mounting medium with DAPI. Then, the slide was observed using a confocal microscope (Zeiss LSM 700, Carl Zeiss, Jena, Germany).

< Example  1> RNase  5 (ribonuclease 5) High expression  Induced person Corneal endothelial cells  Produce

RNase 5 expression plasmid vector was used to induce high expression of RNase 5 in cultured human corneal endothelial cells and the expression level was confirmed (RNase 5 expression plasmid vector injection cultured human corneal endothelial cells are named R5-HCECs).

As a result, mRNA expression of RNase 5 was amplified in cells treated with 0.2 and 0.5 μg doses of the RNase 5 vector as shown in FIGS. 3E and 3F, and at both 24 and 48 hour treatment concentrations.

In addition, in order to confirm that high expression induction of RNase 5 persists after passage, the vector was treated with corneal endothelial cells for 48 hours and stored in culture for 72 hours after passage (pre-passage vector injection corneal endothelial cells were t-P0). , The vector injected corneal endothelial cells after passage are called t-P1). As a result, as shown in FIGS. 3C to 3F, it was confirmed that high expression of both RNase 5 mRNA and protein persisted in t-P1 in RNase 5 vector-injected corneal endothelial cells. On the other hand, in the negative control vector injection corneal endothelial cells (negative control; NC-HCECS) or Lipofectamine 3000 alone, corneal endothelial cells (mock group) did not show a change in expression of RNase 5.

As shown in FIGS. 4A and 4B, in R5-HCEC treated with RNase 5 vector (0.5 μg) for 48 hours, high expression of intracellular RNase 5 protein was observed in the nucleus or in the cytoplasm around the nucleus, but NC-HCEC in the cell RNase 5 was rarely expressed.

Referring to FIG. 4C, it was confirmed that several R5-HCECs simultaneously express RNase 5 in the nucleus and in the periplasm of the nucleus, and the result shows that RNase 5 generated in the cytoplasm translocates into the nucleus of R5-HCEC. Can be suggested as evidence.

< Example  2> cultured person Of corneal endothelial cells  Survival RNase  5's High expression

As the result of survival and growth mechanism activation in cultured human corneal endothelial cells after exogenous human recombinant RNase 5 was externally administered, whether or not manipulation of the expression level of RNase 5 affects the survival of human corneal endothelial cells To evaluate, cell viability was confirmed by performing an MTT assay.

As a result, there was no change in viability at the time point (Day 0, D0) at the moment of passage after injecting 0.2 and 0.5 μg RNase 5 vectors for 48 hours as shown in FIG. 5A. However, referring to FIGS. 5B to 5G, since 24 hours (D1), the viability of R5-HCEC was higher than that of the mock group under simple culture, and it was confirmed that this effect was maintained until Day 6.

< Example  3> RNase  5's In high expression  Cultured by induced In corneal endothelial cells  Cell division-related cell growth changes

The best way to treat nondegenerative human corneal endothelial disease is to proliferate the remaining healthy corneal endothelial cells or supply proliferating human corneal endothelial cells to replace diseased or dead cells. As a matter of the former, the present inventors have previously applied RNase 5 eye drops to the corneal endothelial of frozen-injured rabbit eye, and confirmed that exogenous RNase 5 has the potential to activate cell division of cultured human corneal endothelium. However, in general, medical treatment is usually effective only in mild or moderate cases, whereas severe corneal endothelial damage is inevitably required to be replaced with healthy corneal endothelial cells. Since cell corneal endothelial tissue was quickly filled with normal cells, the cell division-related cell growth of R5-HCEC was evaluated and compared with control corneal endothelial cells.

 First, as a result of performing the BrdU assay as shown in FIG. 6A, it was confirmed that cell division of R5-HCEC (0.2 μg and 0.5 μg, 48 hour treatment) was increased compared to the mock group in the t-P1 passage.

Afterwards, it was confirmed that the high expression of RNase 5 was consequently related to the cell growth rate of HCEC. According to the daily cell growth curve of the control HCEC and R5-HCEC (0.5 μg vector 48 hour treatment) as shown in FIG. 6B, the growth of R5-HCEC was higher for 1 to 3 days compared to the cells of the control cells and the mock group.

From the results, it was confirmed that RNase 5, which is inherently present, is an important substance involved in the proliferation of human corneal endothelial cells.

< Example  4> cultured person Intracorneal cells  of mine RNase  5's High Expression miR -21, 23a and 27a signal mechanism association check

The ribonuclease activity of RNase 5 is essential for biological activity, as the name of RNase 5 itself implies. Although the present inventors have partially revealed that recombinant RNase 5 externally administered to human corneal endothelial cells and fibroblast cells activates the PI3-k / Akt signaling mechanism, the target of endogenous RNase 5 is revealed and detailed ribonucleic acid of RNase 5 Further exploration is still necessary to identify the decomposition mechanism. Micro RNA (miRNA, synonym for miR) is a short (18-25 nucleotide size), endogenous non-coding RNA that inhibits functional RNA transcription and regulates gene expression after transcription. Drosha, an RNase 3 domain enzyme, cuts RNA in the nucleus of the cell and releases the hairpin form of primary micro RNA to produce precursor micro RNA, and further, precursor micro RNA is released into the cytoplasm and is cut by the RNase 3 enzyme Dicer, the final step Becomes the mature micro RNA.

In this context, the present inventors hypothesized that RNase 5 also mimics ribonuclease in the production of micro RNA, and miR-21, miR-, which are known to be associated with cell cycle and cell proliferation among various types of micro RNA. 23a and miR-27a were selected as candidates for the target micro RNA of RNase 5 in R5-HCEC.

 The miR-21 and miR-23a signaling pathways are known to inhibit PTEN, which inhibits PI3-k signaling, and phosphorylate rh Akt to promote cell proliferation, differentiation and motility. In addition, the recently known role of miR-27a is known to contribute to cell proliferation, survival and inhibition of apoptosis).

As a result, as shown in FIG. 7, MiR-21, 23a and 27a in R5-HCEC had higher expression than the mock group. Interestingly, R5-HCEC did not result in miRNA signal changes in the t-P0 passage before passage. In addition, externally administered recombinant RNase 5 (rRNase 5, 1 μg / mL, treated for 24 hours) did not induce changes in miR-21 and 23a, and showed a different pattern from R5-HCECs which increased the expression of endogenous RNase 5 Did.

< Example  5> People Intracorneal cells  Within RNase  5's target  analysis

Among the sub-factors of miR-21 and miR-23a in various studies, substances related to cell proliferation and survival were included, including PDCD4, PTEN, Cdc25A, CdksAP1, Spry1, Spry2 and Apaf1. Briefly, PTEN is a potent inhibitor of the PKB / Akt pathway, and the deletion of PDCD4 is known to be associated with cell proliferation and anti-apoptotic. Cdc25A is a substance required to pass from the G1 phase to the S phase of the cell cycle, and Cdk2AP1 inhibits the action of causing Cdk2 to cross the G1 / S checkpoint of the cell cycle. Active Spry1 is known to promote EGF / EGFR signaling, and Apaf1 acts as a major hub for apoptosis regulatory networks that trigger caspase-3 activation. The present inventors screened the gene expression in the cultured human corneal endothelial cells of these targets and tried to investigate the correlation with RNase 5 overexpression.

As a result, PDCD4 was inhibited in R5-HCEC (0.5 μg vector, 48 hour treatment) among several targets as shown in FIGS. 8A and 8G, and Cdc25A and Apaf1 were activated. On the other hand, PTEN, Cdk2AP1, Spry1 and Spry2 were not significantly affected by RNase 5 overexpression. In addition, as shown in Figure 8H, the cell cycle-related factors Cyclin A2, cyclin D1 and cyclin E1 protein expression were activated in R5-HCEC (0.5 μg vector, 48 hour treatment), and the expression of Cyclin D3 did not increase.

< Example  6> Functional human cornea Endothelial cells  Cell surface to distinguish and evaluate their cell-specific phenotype Marker  Expression analysis

For the innate pump function of the corneal endothelium, the abundant expression of functional markers such as Na ⁺ -K ⁺ ATPase and zonular occludens (ZO) -1 maintains sufficient cell density in vivo and is a very important problem in corneal endothelial cells. to be. In this regard, several researchers have reported that the expression of several CD antigens is associated with the intrinsic phenotype of corneal endothelial cells and are currently using these CD markers to prepare a purified corneal endothelial cell cohort for transplantation. The present inventors evaluated the expression levels of CD44 and CD166, which are representative markers of fibroblastic and non-fibroblastic phenotype, respectively, together with Na ⁺ -K ⁺ ATPase and ZO-1, and confirmed whether they are associated with overexpression of RNase 5, respectively. .

As a result, referring to FIG. 9A, the corneal endothelial cells used in the present invention are 20-year-old female donors, and the shape of the cells in the initial culture showed a medium degree between the shape of the corneal endothelial and the fibroblastic shape. In addition, the corneal endothelial cells in passage 4 were mainly CD44 ⁺ CD166 ⁺ after repeated passages as shown in FIG. 9B, and the fraction of CD44 ⁺ CD166 ⁺ cells was different from R5-HCEC (0.5 μg vector, 48 hour treatment). Did.

In addition, referring to FIGS. 9Ci and 9Cii, both control corneal endothelial cells (passage 4) and R5-HCEC (t-P1) did not exhibit the unique cell phenotype of typical corneal endothelial cells, but R5-as shown in FIGS. 9Ciii to 9Cx. The cell shape of HCEC was less fibroblast compared to control corneal endothelial cells, and the expression of Na ⁺ -K ⁺ ATPase was more pronounced.

Similarly, referring to Figures 9D to 9H, the expression of Na ⁺ -K ⁺ ATPase at the gene and protein levels was also apparent in R5-HCEC (0.5 μg vector, 48 hour treatment), although insignificant, ZO- 1 expression genes TJP1 and CD166 expression genes ALCAM also increased expression in R5-HCEC, and CD44 expression was not significantly related to high expression of RNase 5.

< Example  7> Corneal endothelium made by 3D printer Implant  evaluation

10 days after fabrication of a corneal endothelial implant with a 3D printer and before surgical implantation, the transparency of the implant, the number of cells, the shape of the cells, and the functional phenotype of corneal endothelial cells on the implant were analyzed.

First, as shown in FIG. 10A, it was confirmed that the implant tissue stored in the culture medium for 10 days before surgery maintained its transparency. Each R5-graft (named the implant made by 3D printing R5-HCEC) exhibited a thickness of approximately 20-30 μm, and 3D-printed R5-HCEC cells were between the cell and the support cell-free small membrane. There was one layer with no excitement.

10 days after printing, it was confirmed that corneal endothelial cells formed a unique hexagonal cell shape as shown in FIGS. 10B and 10C. As a result of analyzing the cell number of corneal endothelial cells tracked with CellTracker ™ Green CMFDA dye at the time of 3D printing, the result was analyzed 10 days after printing. As shown in FIG. A 2.5-fold higher number of cells was observed, and the morphology of the cells was also much closer to that of the corneal endothelial cells that were much more unique in R5-HCEC.

In addition, referring to FIG. 11B, the immunofluorescence of Na ⁺ -K ⁺ ATPase and ZO-1 was much more clearly seen in R5-HCEC on R5-graft than control-graft.

< Example  8> Corneal endothelium made by 3D printer Implant  Confirmation of regeneration effect of corneal endothelium in vivo following transplantation

After removing the desmemak into a 8.0-mm diameter circle, a circular graft (R5-graft, control-graft or acellular vesicle) was implanted in a similar manner to DSEK (Descemet's Stripping Endothelial Keratoplasty). In addition, R5-HCEC cells cultured in additional groups were injected into the anterior chamber, and all these experimental groups were compared to a group (control) in which no manipulations such as implantation of implants or cell implantation were performed after desmemak separation.

As a result, severe corneal edema was observed in all eyes after 1 week of surgery as shown in FIGS. Also, referring to FIG. 12Aii, the cells of the cell injection group were accompanied by new blood vessels on the surface of the cornea as seen in advanced blister keratopathy.

On the other hand, referring to Figure 12Axx, the transparency of the cornea began to improve from the second week in the eye of the R5-graft transplantation group, and after 4 weeks of surgery, the transparency of the cornea was almost completely restored and no special inflammatory substance was observed in the anterior chamber. Did. Although the control-graft group showed some improvement in corneal clarity after 4 weeks of surgery, it was not as much as the R5-graft group under microscopic observation.

Also, referring to FIGS. 12Axiii and 12Axviii, both the R5-implant and the control-implant were well attached while the graft remained circular to the cornea during the course of the observation, but the amnion implanted in the group transplanted with the cell-free small membrane was performed. It began to contract after 3 weeks.

Meanwhile, as a result of measuring the central corneal thickness (CCT) reflecting the degree of corneal edema, severe and persistent corneal edema was observed in the control group, cell infusion group, and cell-free amniotic membrane transplantation group by 2 weeks. There was a slight decrease in corneal thickness at 3-4 weeks in the cell injection group, but there was no statistically significant difference between the cell injection group, the cell-free amniotic membrane transplantation group and the control group. However, in the control-implant and R5-implant group, the central corneal thickness gradually began to decrease after 2 weeks of surgery, and at 3 and 4 weeks, the thickness was significantly lower than that of the control group. -There was no statistically significant difference between the graft group and the R5-graft group, and no eye showed an increase in intraocular pressure at 4 weeks.

As a result of histological observation and analysis of corneal endothelium in 3 eyes of the R5-graft group 4 weeks after surgery, the rear part of the transplanted R5-graft was filled with a single layer of cells with no gaps, and the average density of the cells was 3,073 ± 341.2 (Mean ± standard error) cells / mm ² . In addition, as a result of observing anti-dye fluorescence under a microscope, as shown in FIG. 12E, these were not corneal endothelial cells of rabbits, but human corneal endothelial cells transplanted by culture were identified, and as shown in FIG. Markers such as Na ⁺ -K ₊ ATPase, ZO-1 and CD166 are clearly expressed along the hexagonal border of the cell, whereas expression of Na ⁺ -K ⁺ ATPase in the corneal endothelial cells of the control-graft is R5-graft. Contrast was relatively low.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Preparing method for 3 dimension corneal endothelial graft          comprising ribonuclease 5 overexepressing corneal endothelial          cells <130> ADP-2018-0032 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 147 <212> PRT <213> ribonuclease 5 <400> 1 Met Val Met Gly Leu Gly Val Leu Leu Leu Val Phe Val Leu Gly Leu   1 5 10 15 Gly Leu Thr Pro Pro Thr Leu Ala Gln Asp Asn Ser Arg Tyr Thr His              20 25 30 Phe Leu Thr Gln His Tyr Asp Ala Lys Pro Gln Gly Arg Asp Asp Arg          35 40 45 Tyr Cys Glu Ser Ile Met Arg Arg Arg Gly Leu Thr Ser Pro Cys Lys      50 55 60 Asp Ile Asn Thr Phe Ile His Gly Asn Lys Arg Ser Ile Lys Ala Ile  65 70 75 80 Cys Glu Asn Lys Asn Gly Asn Pro His Arg Glu Asn Leu Arg Ile Ser                  85 90 95 Lys Ser Ser Phe Gln Val Thr Thr Cys Lys Leu His Gly Gly Ser Pro             100 105 110 Trp Pro Pro Cys Gln Tyr Arg Ala Thr Ala Gly Phe Arg Asn Val Val         115 120 125 Val Ala Cys Glu Asn Gly Leu Pro Val His Leu Asp Gln Ser Ile Phe     130 135 140 Arg Arg Pro 145

Claims (11)

Overexpressing ribonuclease 5 on corneal endothelial cells (first step);
Dispersing corneal endothelial cells overexpressed in the first step of ribonuclease 5 in bioink (second step);
Separating the bioink in which the corneal endothelial cells of the second step are dispersed from livestock and bioprinting the lyophilized amniotic membrane (third step); And
A method of manufacturing a 3D bioprinted corneal endothelial implant comprising the step of irradiating ultraviolet rays to the bioprinted amniotic membrane of the third step to cross-link them (the fourth step). The method according to claim 1, wherein the first step of the ribonuclease 5 overexpressed corneal endothelial cells are transformed corneal endothelial cells with a recombinant vector containing the ribonuclease 5 gene encoding the amino acid sequence represented by SEQ ID NO: 1 Method for producing a corneal endothelial implant characterized by overexpressing ribonuclease 5 by conversion. The method according to claim 2, wherein the recombinant vector is a corneal endothelial implant manufacturing method, characterized in that the following cleavage map 1.
[Open map 1]

The method of claim 1, wherein the corneal endothelial cells are corneal endothelial cells separated from humans. The method according to claim 1, wherein the bioink in which the corneal endothelial cells are dispersed in the second step is characterized in that the corneal endothelial cells overexpressing ribonuclease 5 are dispersed in bioink at a concentration of 3 × 10 ⁶ cells / mL. Method for manufacturing an endothelial implant. The method according to claim 1, wherein the third step is a method for producing a corneal endothelial implant, characterized in that bioprinting of the corneal endothelial cells dispersed in the lyophilized and acellularized bovine membrane is 0.5 to 1 mm thick. The method according to claim 1, wherein the fourth step is a method of manufacturing a corneal endothelial implant, characterized by cross-linking the bioprinted amniotic membrane by irradiating 360 nm ultraviolet rays for 10 to 20 seconds. The method according to claim 1, wherein the method of manufacturing a corneal endothelial implant further comprises the step of culturing the corneal endothelial implant irradiated with ultraviolet rays after the fourth step at 37 ° C for 10 days. A corneal endothelial implant in which corneal endothelial cells overexpressing ribonuclease 5 according to any one of claims 1 to 8 are 3D bioprinted on the amniotic membrane. The method according to claim 9, wherein the corneal endothelial implant is a 3D bioprinted ribonuclease 5 (Ribonuclease 5) overexpressed on the amniotic membrane, the corneal endothelial cells 3D bioprinted ribonuclease 5 on the amniotic membrane is not overexpressed. A corneal endothelial implant characterized by increased expression of Na ⁺ -K ⁺ ATPase and CD166 than control corneal endothelial cells. The method according to claim 9, wherein the corneal endothelial implant is a three-dimensional bioprinted ribonuclease 5 (Ribonuclease 5) overexpressed on the amniotic membrane, the corneal endothelial cells are three-dimensional bioprinted ribonuclease 5 on the amniotic membrane is not overexpressed. A corneal endothelial implant, characterized in that the expression of zonular occludens-1 (ZO-1) is increased compared to the control corneal endothelial cells, thereby increasing the corneal endothelial phenotype.

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