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Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro.

Ma X, Lin Y, Yang K, Yue B, Xiang H, Chen B - Int. J. Mol. Med. (2015)

Bottom Line: The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV‑mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage.Furthermore, no significant anti-apoptotic effects were observed following LV‑mediated survivin transfection.However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University Medical College, Qingdao University, Qingdao, Shandong 266003, P.R. China.

ABSTRACT
Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)‑mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV‑mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT‑qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase‑3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV‑mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase‑3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV‑mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive studies on the feasibility of using survivin in gene therapy in an aim to attenuate disc degeneration are warranted. Further research on the mechanisms responsible for the changes in cell morphology and cell function are also required.

No MeSH data available.


Related in: MedlinePlus

Isolation, culture and identification of primary nucleus pulposus (NP) cells. (A) Primary NP cells were obtained by enzymatic digestion (magnification, ×100). (B) Degenerated NP cells from passage 2 attached to the culture dish after passage for 3 h (magnification, ×100). The primary NP cells derived from degenerative human disc were round at the moment of isolation (A) and they had attached to the culture dish after 5–7 days of culture. The cells gradually became elongated and triangular or polygonal in shape, and the cytoplasm became plump and equally distributed. The number of attached cells exponentially increased. (C and D) Immunostaining revealed that these cells were positive for aggrecan and type II collagen, which are makers of the NP cell phenotype (magnification, ×100).
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f3-ijmm-36-01-0186: Isolation, culture and identification of primary nucleus pulposus (NP) cells. (A) Primary NP cells were obtained by enzymatic digestion (magnification, ×100). (B) Degenerated NP cells from passage 2 attached to the culture dish after passage for 3 h (magnification, ×100). The primary NP cells derived from degenerative human disc were round at the moment of isolation (A) and they had attached to the culture dish after 5–7 days of culture. The cells gradually became elongated and triangular or polygonal in shape, and the cytoplasm became plump and equally distributed. The number of attached cells exponentially increased. (C and D) Immunostaining revealed that these cells were positive for aggrecan and type II collagen, which are makers of the NP cell phenotype (magnification, ×100).

Mentions: The primary NP cells derived from degenerative human disc were round at the moment of isolation (Fig. 3A) and they had attached to the culture dish after 5–7 days of culture. The cells gradually became elongated and triangular or polygonal in shape, and the cytoplasm became plump and equally distributed. The number of attached cells exponentially increased. After 15–20 days, 90% of the cells had formed colonies. The passaged NP cells derived from degenerative human disc only required 3 h to attach to the culture dish, and 90% of the cells formed colonies after 7–10 days of culture. Cell morphology was similar to that of primary cells (Fig. 3B).


Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro.

Ma X, Lin Y, Yang K, Yue B, Xiang H, Chen B - Int. J. Mol. Med. (2015)

Isolation, culture and identification of primary nucleus pulposus (NP) cells. (A) Primary NP cells were obtained by enzymatic digestion (magnification, ×100). (B) Degenerated NP cells from passage 2 attached to the culture dish after passage for 3 h (magnification, ×100). The primary NP cells derived from degenerative human disc were round at the moment of isolation (A) and they had attached to the culture dish after 5–7 days of culture. The cells gradually became elongated and triangular or polygonal in shape, and the cytoplasm became plump and equally distributed. The number of attached cells exponentially increased. (C and D) Immunostaining revealed that these cells were positive for aggrecan and type II collagen, which are makers of the NP cell phenotype (magnification, ×100).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494593&req=5

f3-ijmm-36-01-0186: Isolation, culture and identification of primary nucleus pulposus (NP) cells. (A) Primary NP cells were obtained by enzymatic digestion (magnification, ×100). (B) Degenerated NP cells from passage 2 attached to the culture dish after passage for 3 h (magnification, ×100). The primary NP cells derived from degenerative human disc were round at the moment of isolation (A) and they had attached to the culture dish after 5–7 days of culture. The cells gradually became elongated and triangular or polygonal in shape, and the cytoplasm became plump and equally distributed. The number of attached cells exponentially increased. (C and D) Immunostaining revealed that these cells were positive for aggrecan and type II collagen, which are makers of the NP cell phenotype (magnification, ×100).
Mentions: The primary NP cells derived from degenerative human disc were round at the moment of isolation (Fig. 3A) and they had attached to the culture dish after 5–7 days of culture. The cells gradually became elongated and triangular or polygonal in shape, and the cytoplasm became plump and equally distributed. The number of attached cells exponentially increased. After 15–20 days, 90% of the cells had formed colonies. The passaged NP cells derived from degenerative human disc only required 3 h to attach to the culture dish, and 90% of the cells formed colonies after 7–10 days of culture. Cell morphology was similar to that of primary cells (Fig. 3B).

Bottom Line: The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV‑mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage.Furthermore, no significant anti-apoptotic effects were observed following LV‑mediated survivin transfection.However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University Medical College, Qingdao University, Qingdao, Shandong 266003, P.R. China.

ABSTRACT
Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)‑mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV‑mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT‑qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase‑3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV‑mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase‑3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV‑mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive studies on the feasibility of using survivin in gene therapy in an aim to attenuate disc degeneration are warranted. Further research on the mechanisms responsible for the changes in cell morphology and cell function are also required.

No MeSH data available.


Related in: MedlinePlus